Fibrinolytic and anti-thrombotic cleavable dimers

ABSTRACT

Relatively inactive fusion proteins are activatable by enzymes of the clotting cascade to have fibrinolytic and/or clot formation inhibition activity. For example, a fusion protein comprising two hirudin or streptokinase molecules, linked by a cleavable linkage sequence, may be cleaved to yield anti-thrombotic hirudin or fibrinolytic streptokinase by thrombin or Factor Xa. Fibrinolytic or clot formation inhibition activity is therefore directed to the site of clot formation. Cleavable streptokinase/hirudin heterodimers are claimed.

This application is the US national stage under 37 CFR 371 ofPCT/GB90/01911, filed 7 Dec. 1990.

This invention relates to proteinaceous compounds which can be cleavedto release fibrinolytic and/or anti-thrombotic activity. It also relatesto nucleic acid (DNA and RNA) coding for all or part of such compounds.In preferred embodiments, the invention relates to fusion proteinsproduced by linking together fibrinolytic and/or anti-thromboticproteins with a cleavable linker, their preparation, pharmaceuticalcompositions containing them and their use in the treatment ofthrombotic disease.

The fibrinolytic system is the natural counterpart to the clottingsystem in the blood. In the process of blood coagulation, a cascade ofenzyme activities are involved in generating a fibrin network whichforms the framework of a clot, or thrombus. Degradation of the fibrinnetwork (fibrinolysis) is accomplished by the action of the enzymeplasmin. Plasminogen is the inactive precursor of plasmin and conversionof plasminogen to plasmin is accomplished by cleavage of the peptidebond between arginine 561 and valine 562 of plasminogen. Underphysiological conditions this cleavage is catalysed by tissue-typeplasminogen activator (tPA) or by urokinase-type plasminogen activator(uPA).

If the balance between the clotting and fibrinolytic systems becomeslocally disturbed, intravascular clots may form at inappropriatelocations leading to conditions such as coronary thrombosis andmyocardial infarction, deep vein thrombosis, stroke, peripheral arterialocclusion and embolism. In such cases, the administration offibrinolytic and anti-thrombotic agents has been shown to be abeneficial therapy for the promotion of clot dissolution.

Fibrinolytic therapy has become relatively widespread with theavailability of a number of plasminogen activators such as tPA, uPA,streptokinase and the anisoylated plasminogen streptokinase activatorcomplex, APSAC. Each of these agents has been shown to promote clotlysis, but all have deficiencies in their activity profile which makesthem less than ideal as therapeutic agents for the treatment ofthrombosis (reviewed by Marder and Sherry, New England Journal ofMedicine 1989, 318: 1513-1520).

A major problem shared by all of these agents is that at clinicallyuseful doses, they are not thrombus specific as they activateplasminogen in the general circulation. The principal consequence ofthis is that proteins such as fibrinogen involved in blood clotting aredestroyed and dangerous bleeding can occur. This also occurs with tPAdespite the fact that, at physiological concentrations, it binds tofibrin and shows fibrin selective plasminogen activation.

Another important shortcoming in the performance of existing plasminogenactivators is that re-occlusion of the reperfused blood vessel commonlyoccurs after cessation of administration of the thrombolytic agent. Thisis thought to be due to the persistence of thrombogenic material at thesite of thrombus dissolution. Anti-thrombotic proteins may be used inthe treatment or prophylaxis of thrombosis either alone or as an adjunctto fibrinolytic agents. Suitable anti-thrombotic proteins includehirudin, activated protein C and anti-thrombin III.

An alternative approach to enhancing fibrinolysis and inhibition ofblood clotting has now been devised which is based on the use of fusionproteins cleavable to achieve release of fibrinolytic and/oranti-thrombotic activity at the site of blood clotting. To achieve this,proteins involved in fibrinolysis or inhibition of coagulation arejoined by a linker region which is cleavable by an enzyme involved inblood clotting. Examples of proteins which may be incorporated into sucha cleavable protein include tPA, uPA, streptokinase, plasminogen,activated protein C, hirudin and anti-thrombin III. Fusion of suchproteins to a protein with a favourable property not directly related todissolution of blood clots, for example albumin which has a long plasmahalf-life, may also be beneficial. An advantage of this approach is thatthrombus selectivity of fibrinolytic or inhibition of clot formationactivity is achieved by way of the thrombus-specific localisation of thecleaving enzymes.

According to a first aspect of the invention, there is provided a fusionprotein comprising a first sequence and a second sequence, the fusionprotein being cleavable between the first and second sequences by anenzyme involved in blood clotting, wherein after the fusion protein isso cleaved the first and second sequences, or either of them, hasgreater fibrinolytic and/or anti-thrombotic activity than the uncleavedfusion protein.

The fusion protein may be a cleavable dimer of two fibrinolytic and/oranti-thrombotic proteins, such as hirudin or streptokinase. It may be ahomodimer or a heterodimer. The fusion protein may have substantiallyreduced or no fibrinolytic and/or anti-thrombotic activity compared tothe cleavage products, but a certain amount of activity in the fusionprotein can be tolerated. It is not necessary for both the cleavageproducts to have fibrinolytic and/or anti-thrombotic activity, but it ispreferred for them to do so.

The fusion protein is not restricted to being a dimer; it may have anynumber (such as three, four or more) sequences which are cleavable onefrom the other, compatible with the therapeutic utility of the protein.At least one, and preferably more than one or even all, of the sequencesresulting from the cleavage will have greater activity than the fusionprotein, or a combination of some or all of the cleavage products willcollectively have such greater activity. In any event, cleavage willresult in a net increase in or release of activity.

Proteinaceous compounds in accordance with the first aspect of theinvention, are therefore cleaved to release activity in at least one oftwo ways. First, a compound may be cleaved to release fibrinolyticactivity. Secondly, a compound may be cleaved to release anti-thromboticactivity. Conceivably, a compound may be cleaved to release bothfunctions. It should be noted that a released fragment of the fusionprotein may have fibrinolytic activity directly (in that it lysesfibrin) or indirectly (in that it causes activation of a molecule whichleads to lysis of fibrin).

One preferred proteinaceous compound which is cleavable to have enhancedanti-thrombotic activity is a fusion protein of two hirudin moleculeslinked (for example carboxy terminus to amino terminus) by a linkeramino acid sequence cleavable, for example, by Factor Xa.

Hirudins are naturally occurring polypeptides of 65 or 66 amino acids inlength that are produced by the leech Hirudo medicinalis. Hirudin is ananticoagulating agent which binds to thrombin and prevents bloodcoagulation by inhibiting thrombin from catalysing the conversion offibrinogen to fibrin, thus preventing the formation of the proteinframework of blood clots. The binding of hirudin also prevents otherprothrombic activities of thrombin, including activation of factors V,VII, XIII and platelets. There are three principal variants of hirudin(named HV-1, HV-2 and HV-3).

Another preferred fusion protein comprises two streptokinase moleculeslinked (for example carboxy terminus to amino terminus) by a linkeramino acid sequence cleavable, for example, by thrombin.

Streptokinase is a 414 amino acid, 47 kDa protein secreted by manypathogenic streptococci of different serogroups. It is a plasminogenactivator but, unlike mammalian plasminogen activators, it is not aprotease and it activates plasminogen by forming a binary complex withplasminogen (SK-plasminogen) which functions as an activator of freeplasminogen. Streptokinase is effective in inducing clot lysis in thetreatment of myocardial infarction and is widely used for thisindication.

Cleavable fusion proteins within the scope of this invention may havereduced fibrinolytic and/or anti-thrombotic activity compared to theircomponent molecules; cleavage releases the component molecules whichpossess to an adequate degree the activity of their wild-type parentmolecules.

The blood coagulation mechanism comprises a series of enzyme reactionswhich culminate in the production of insoluble fibrin, which forms themesh-like protein framework of blood clots. Thrombin is the enzymeresponsible for the conversion of soluble fibrinogen to fibrin.Conversion of prothrombin, the inactive precursor of thrombin, tothrombin is catalysed by activated Factor X (Factor Xa). (Thrombin isalso known as Factor IIa, and prothrombin as Factor II.)

Factor Xa is generated from Factor X extrinsically or intrinsically. Inthe extrinsic route, Factor VII is activated to Factor VIIa, whichgenerates Factor Xa from Factor X. In the intrinsic route, theactivation of Factor X to Factor Xa is catalysed by Factor IXa. FactorIXa is generated from Factor IX by the action of Factor XIa, which inturn is generated by the action of Factor XIIa on Factor XI. Factor XIIais generated from Factor XII by the action of Kallikrein. Factors VIIIaand Va are thought to act as cofactors in the activation of Factors Xand II, respectively.

Fibrin, as first formed from fibrinogen, is in the loose form. Loosefibrin is converted to tight fibrin by the action of Factor XIIIa, whichcrosslinks fibrin molecules.

Activated protein C is an anticoagulant serine protease generated in thearea of clot formation by the action of thrombin, in combination withthrombomodulin, on protein C. Activated protein C regulates clotformation by cleaving and inactivating the pro-coagulant cofactors Vaand VIIIa.

The term "enzyme involved in blood clotting" as used in thisspecification therefore includes kallikrein Factors XIIa, XIa, IXa,VIIa, Xa and thrombin (Factor IIa), which are directly involved in theformation of fibrin and activated protein C, which is involved in thecontrol of blood clotting. The most preferred enzymes are Factor Xa andthrombin because they are most immediately involved with fibrinformation.

Generation and activity of at least Factor Xa and thrombin is tightlyregulated to ensure that thrombus generation is restricted to the siteof the thrombogenic stimulus. This localisation is achieved by thecombined operation of at least two control mechanisms: the bloodclotting enzymes function as complexes intimately associated with thephospholipid cellular membranes of platelets and endothelial cells atthe site of vascular injury (Mann, K. G., 1984, in:

"Progress in Hemostasis and Thrombosis", 1-24, ed Spaet, T. H. Grune andStratton); and, free thrombin or Factor Xa released from the thrombussite into the circulation is rapidly inactivated by the action ofproteinase inhibitors such as anti-thrombin III.

Thus, the activity of the penultimate (Factor Xa) and the final(thrombin) enzymes in the clotting cascade are particularly welllocalised to the site of thrombus generation and for this reason arepreferred. Thrombin has been found to remain associated with thrombi andto bind non-covalently to fibrin. On digestion of thrombi with plasmin,active thrombin is liberated and is thought to contribute to thereformation of thrombi and the re-occlusion of vessels which commonlyoccurs following thrombolytic treatment with plasminogen activators(Bloom A. L., 1962, Br. J. Haematol, 82 129; Francis et al, 1983, J.Lab. Clin. Med., 102, 220; Mirshahi et al, 1989, Blood 74, 1025).

For these reasons, it is preferred in certain embodiments of theinvention to produce fusion proteins activatable by thrombin or FactorXa thereby to create a preferred class of thrombus-selective,fibrinolytic proteins. The most preferred of these fusion proteinsregains the favourable properties of the parent molecules upon cleavageand exhibit thrombus selectivity by the novel property of being cleavedto release the component proteins of the fusion protein at the site ofnew thrombus formation by the action of one of the enzymes involved ingeneration of the thrombus and preferably localised there. Factor Xa(E.C.3.4.21.6) is a serine protease which converts human prothrombin tothrombin by specific cleavage of the Arg(273)-Thr(274) andArg(322)-Ile(323) peptide bonds (Mann et al 1981, Methods in Enzymology80 286-302). In human prothrombin, the Arg(273)-Thr(274) site ispreceded by the tripeptide Ile-Glu-Gly and the Arg(322)-Ile(323) site ispreceded by the tripeptide Ile-Asp-Gly. The structure required forrecognition by Factor Xa appears to be determined by the local aminoacid sequence preceding the cleavage site (Magnusson et al, 1975, in:"Proteases and Biological Control", 123-149 eds., Reich et al, ColdSpring Harbor Laboratory, New York). Specificity for the Ile-Glu-Gly-Argand Ile-Asp-Gly-Arg sequence is not absolute as Factor Xa has been foundto cleave other proteins, for example Factor VIII at positions 336, 372,1689 and 1721, where the preceding amino acid sequence differssignificantly from this format (Eaton et al, 1986 Biochemistry 25505-512). As the principal natural substrate for Factor Xa isprothrombin, preferred recognition sequences are those in which arginineand glycine occupy the P1 and P2 positions, respectively, an acidicresidue (aspartic or glutamic acid) occupies the P3 position andisoleucine or another small hydrophobic residue (such as alanine,valine, leucine or methionine) occupies the P4 position. However, asFactor Xa can cleave sequences which differ from this format, othersequences cleavable by Factor Xa may be used in the invention, as canother sequences cleavable by other enzymes of the clotting cascade.

In order to make fusion proteins which are cleavable by these preferredenzymes, the amino acid sequence linking the components of the fusionprotein must be recognised as a cleavage site for these preferredenzymes. To make fusion proteins which are cleaved by, for example,Factor Xa, an amino acid sequence cleavable by Factor Xa may be used tolink the two components (that is, the first and second, and possiblyother, sequences) of the fusion protein. The sequence Ile-Glu-Gly-ArgSEQ ID NO: 71, which is at one of the sites in prothrombin cleaved byFactor Xa, may be such a sequence. Other possibilities would besequences or mimics of sequences cleaved by Factor Xa in other proteinsor peptides. DNA coding for the Ile-Glu-Gly-Arg SEQ ID NO: 71 sequenceas the carboxy-terminal part of a cleavable linker as a proteinproduction aid is disclosed in UK Patent Application GB-A-2160206 butthe use of an Ile-Glu-Gly-Arg SEQ ID NO: 71 sequence for the purpose ofthis invention is not disclosed in that specification.

Cleavage of fusion proteins by an enzyme of the clotting cascade such asthrombin or Factor Xa can be measured in a number of ways, for exampleby SDS-PAGE analysis, and by assaying for the functions of one or moreof the cleavage products of the fusion protein.

Thrombin (E.C. 3.4.21.5) is a serine protease which catalyses theproteolysis of a number of proteins including fibrinogen (A alpha and Bbeta chains), Factor XIII, Factor V, Factor VII, Factor VIII, protein Cand anti-thrombin III. The structure required for recognition bythrombin appears to be partially determined by the local amino acidsequence around the cleavage site but is also determined to a variableextent by sequence(s) remote from the cleavage site. For example, in thefibrinogen A alpha chain, residues P2 (Val), P9 (Phe) -and P10 (Asp) arecrucial for α-thrombin-catalysed cleavage at the Arg(16)-Gly(17) peptidebond (Ni, F. et al 1989, Biochemistry 28 3082-3094). Comparative studiesof several proteins and peptides which are cleaved by thrombin has ledto the proposal that optimum cleavage sites for α-thrombin may have thestructure of (i) P4-P3-Pro-Arg-P1'-P2'SEQ ID NO: 72, where each of P3and P4 is independently a hydrophobic amino acid (such as valine) andeach of P1' and P2' is independently a non-acidic amino acids, or (ii)P2-Arg-P1' where P2 or P1' is glycine (Chang, J. 1985, Eur. J. Biochem.151 217-224). There are, however, exceptions to these general structureswhich are cleaved by thrombin and which may be used in the invention.

To produce a fusion protein which could be cleaved by thrombin, a linkersequence containing a site recognised and cleaved by thrombin may beused. An amino acid sequence such as that cleaved by thrombin in thefibrinogen A alpha chain may be used. Other possible sequences wouldinclude those involved in the cleavage by thrombin of fibrinogen B beta,Factor XIII, Factor V, Factor VII, Factor VIII, protein C, anti-thrombinIII and other proteins whose cleavage is catalysed by thrombin. Anexample of a thrombin cleavable linker may be the sequence Gly-Pro-Argwhich is identical to that found at positions 17-20 in fibrinogen Aalpha. This is not the principal thrombin cleavage site in fibrinogen Aalpha but thrombin can cleave the Arg(19)-Val(20) peptide bond. Anothersuitable thrombin cleavable linker sequence isVal-Glu-Leu-Gln-Gly-Val-Val-Pro-Arg which is identical to that found inFactor XIII.

In a preferred embodiment the invention relates to fusion proteins ofstreptokinase and/or hirudin linked by peptide sequences which arecleaved by thrombin, Factor Xa or other enzymes involved in bloodclotting to release products with fibrinolytic and/or anti-thromboticactivity.

Fusion proteins in accordance with the invention may contain othermodifications (as compared to wild-type counterparts of their componentssuch as streptokinase and hirudin) which may be one or more additions,deletions or substitutions. An example of such a modification would bestreptokinase variants in which inappropriate glycosylation during yeastexpression was prevented by substitution of sequences recognised asglycosylation signals by yeast. Another example would be the addition ofan Arg-Gly-Asp-Xaa sequence, where Xaa represents a variable amino acidsuch as Ser, to the carboxy terminus of the fusion to enhance its plasmalifetime.

Preferred features of fusion proteins within the scope of the inventionalso apply, where appropriate, to other compounds of the invention,mutatis mutandis.

Fusion proteins in accordance with the first aspect of the invention canbe synthesised by any convenient route. According to a second aspect ofthe invention there is provided a process for the preparation of aproteinaceous compound as described above, the process comprisingcoupling successive amino acid residues together and/or ligatingoligopeptides. Although proteins may in principle be synthesised whollyor partly by chemical means, the route of choice will be ribosomaltranslation, preferably in vivo, of a corresponding nucleic acidsequence. The protein may be glycosylated appropriately.

It is preferred to produce proteins in accordance with the invention byusing recombinant DNA technology. DNA encoding each of the first andsecond sequences of the fusion protein may be from a cDNA or genomicclone or may be synthesised. Amino acid substitutions, additions ordeletions are preferably introduced by site-specific mutagenesis.Suitable DNA sequences encoding streptokinase and hirudin and otherpolypeptide sequences useful in the scope of the invention may beobtained by procedures familiar to those having ordinary skill ingenetic engineering. For several proteins, it is a routine procedure toobtain recombinant protein by inserting the coding sequence into anexpression vector and transfecting or transforming the vector into asuitable host cell. A suitable host may be a bacterium such as E. coli,a eukaryotic microorganism such as yeast or a higher eukaryotic cell.

According to a third aspect of the invention, there is providedsynthetic or recombinant nucleic acid coding for a proteinaceouscompound as described above. The nucleic acid may be RNA or DNA.Preferred characteristics of this aspect of the invention are as for thefirst aspect.

According to a fourth aspect of the invention, there is provided aprocess for the preparation of nucleic acid in accordance with the thirdaspect, the process comprising coupling successive nucleotides togetherand/or ligating oligo- and/or polynucleotides.

Recombinant nucleic acid in accordance with the third aspect of theinvention may be in the form of a vector, which may for example be aplasmid, cosmid or phage. The vector may be adapted to transfect ortransform prokaryotic (for example bacterial) cells and/or eukaryotic(for example yeast or mammalian) cells. A vector will comprise a cloningsite and usually at least one marker gene. An expression vector willhave a promoter operatively linked to the sequence to be inserted intothe cloning site and, preferably, a sequence enabling the proteinproduct to be secreted. Expression vectors and cloning vectors (whichneed not be capable of expression) are included in the scope of theinvention.

It is to be understood that the term "vector" is used in thisspecification in a functional sense and is not to be construed asnecessarily being limited to a single nucleic acid molecule.

Using a vector, for example as described above, fusion proteins inaccordance with the invention may be expressed and secreted into thecell culture medium in a biologically active form without the need forany additional biological or chemical procedures. Suitable cells or celllines to be transformed may be mammalian cells which grow in continuousculture and which can be transfected or otherwise transformed bystandard techniques. Examples of suitable cells include Chinese hamsterovary (CHO) cells, mouse myeloma cell lines such as P3X63-Ag8.653, COScells, HeLa cells, BHK cells, melanoma cell lines such as the Bowes cellline, mouse L cells, human hepatoma cell lines such as Hep G2, mousefibroblasts and mouse NIH 3T3 cells. Such cells may be particularlyappropriate for expression when one or more of the protein sequencesconstituting the fusion protein is of mammalian derivation, such astissue plasminogen activator (t-PA).

Yeast (for example Pichia pastoris or Saccharomyces cerevisiae) orbacteria (for example Escherichia coli) may be preferred for theexpression of many of the fusion proteins of the invention, as mayinsect cells such as those which are Baculovirus-infected.

Compounds of the present invention may be used within pharmaceuticalcompositions for the prevention or treatment of thrombosis or otherconditions where it is desired to produce local fibrinolytic and/oranticoagulant activity. Such conditions include myocardial and cerebralinfarction, arterial and venous thrombosis, thromboembolism,post-surgical adhesions, thrombophlebitis and diabetic vasculopathies.

According to a fifth aspect of the invention, there is provided apharmaceutical composition comprising one or more compounds inaccordance with the first aspect of the invention and a pharmaceuticallyor veterinarily acceptable carrier. Such a composition may be adaptedfor intravenous administration and may thus be sterile. Examples ofcompositions in accordance with the invention include preparations ofsterile fusion proteins in isotonic physiological saline and/or buffer.The composition may include a local anaesthetic to alleviate the pain ofinjection. Compounds of the invention may be supplied in unit dosageform, for example as a dry powder or water-free concentrate in ahermetically sealed container such as an ampoule or sachet indicatingthe quantity of protein. Where a compound is to be administered byinfusion, it may be dispensed by means of an infusion bottle containingsterile water for injections or saline or a suitable buffer. Where it isto be administered by injections, it may be dispensed with an ampoule ofwater for injection, saline or a suitable buffer. The infusible orinjectable composition may be made up by mixing the ingredients prior toadministration. Where it is to be administered as a topical treatment,it may be dispensed in a suitable base.

The quantity of material to be administered will depend on the amount offibrinolysis or inhibition of clotting required, the required speed ofaction, the seriousness of the thromboembolic position and the size ofthe clot. The precise dose to be administered will, because of the verynature of the condition which compounds of the invention are intended totreat, be determined by the physician. As a guideline, however, apatient being treated for a mature thrombus will generally receive adaily dose of a fusion protein of from 0.01 to 10 mg/kg of body weighteither by injection in for example up to 5 doses or by infusion.

The invention may be used in a method for the treatment or prophylaxisof thrombosis, comprising the administration of an effective non-toxicamount of a compound in accordance with the first aspect. According to afurther aspect of the invention, there is therefore provided the use ofa compound as described above in the preparation of a thrombolyticand/or anticoagulant agent.

The invention concerns especially the DNAs, the vectors, the transformedhost strains, the fusion proteins and the process for the preparationthereof as described in the examples.

The following examples of the invention are offered by way ofillustration, and not by way of limitation. The examples refer to theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically the arrangement of a set of oligonucleotidesused in the assembly of a synthetic hirudin gene (Preparation 1);

FIG. 2 shows a map of plasmid pSW6 (Preparation 2);

FIG. 3 shows a map of plasmid pJK1 (Preparation 2);

FIG. 4 shows a map of plasmid pGC517 (Example 4);

FIG. 5 shows a zymograph of E. coli strains expressing streptokinaseactivity (Example 11); and

FIG. 6 shows a zymograph demonstrating cleavage of astreptokinase-streptokinase fusion protein by thrombin (Example 13).

FIG. 7 shows complete nucleotide sequences corresponding to theschematic arrangement depicted in FIG. 1.

METHODOLOGY

The techniques of genetic engineering and genetic manipulation used inthe manufacture of the genes described and in their further manipulationfor construction of expression vectors are well known to those skilledin the art. Descriptions of modern techniques can be found in thelaboratory manuals "Current Protocols in Molecular Biology", Volumes 7and 2, edited by F. M. Ausubel et al, published by Wiley-Interscience,New York and in "Molecular Cloning, A Laboratory Manual" (secondedition) edited by Sambrook, Fritsch and Maniatis published by ColdSpring Harbor Laboratories, New York. M13mp18, M13mp19 and pUC19 DNAswere purchased from Pharmacia Ltd., Midsummer Boulevard, Central MiltonKeynes, Bucks, MK9 3HP, United Kingdom. Restriction endonucleases werepurchased either from Northumbria Biologicals Limited, South NelsonIndustrial Estate, Cramlington, Northumberland, NE23 9HL, United Kingdomor from New England Biolabs, 32 Tozer Road, Beverly, Mass. 01915-5510USA. E. coli HW1110 (lacIq) is used as expression host in certain of thefollowing examples: a suitable commercially available alternative isJM109, available from Northumbria Biologicals Ltd.

PREPARATION 1 Construction of a Hirudin HV1 Gene A. Gene Design

A synthetic hirudin HV-1 gene was designed based on the published aminoacid sequence (Dodt J., et al FEBS Letters 65 180 (1984)). Uniquerestriction endonuclease target sites were incorporated to facilitatesubsequent genetic manipulation (see SEQ. ID NO:1 and 2 in the SequenceListings immediately before the claims). The codons selected were thosefavoured by either S. cerevisiae or E. coli and are thus suitable forexpression in either organism.

B. Gene Construction

The gene sequence was divided into 12 oligodeoxyribonucleotides (seeSEQ. ID NOS: 50 through 61) such that after annealing each complementarypair 2 oligonucleotides, they were left with cohesive ends either for orof 7 bases in length.

C. Oligonucleotide Synthesis

The oligonucleotides were synthesised by automated phosphoramiditechemistry on an Applied Bio-Systems 380B DNA Synthesiser, usingcyanoethyl phosphoramidites. The methodology is now widely used and hasalready been described (Beaucage, S. L. and Caruthers, M. H. TetrahedronLetters 24, 245 (1981) and Caruthers, M. H. Science 230, 281-285(1985)).

D. Gene Assembly

The oligonucleotides were kinased to provide them with a 5' phosphate toallow their subsequent ligation. The oligonucleotides were assembled asshown in FIGS. 1 and 7.

Kinasing of Oligomers

100 pmole of oligomer was dried down and resuspended in 20 μl kinasebuffer (70 mM Tris, pH 7.6, 10 mM MgCl₂, 1 mM ATP, 0.2 mM spermidine,0.5 mM dithiothreitol (DTT)). T4 polynucleotide kinase (2 mcl. 10 000U/ml) was added and the mixture was incubated at 37° C. for 30 minutes.The kinase was then inactivated by heating at 70° C for 10 minutes.

Complementary pairs of kinased oligonucleotides were annealed in pairs(90° C., 5 minutes, followed by slow cooling at room temperature). The 6paired oligomers were then mixed together, incubated at 50 ° C. for 5minutes and allowed to cool. They were then ligated overnight at 16° C.with T4 DNA ligase. The strategy is shown diagrammatically in FIGS. 1and 7 (note P=5'-phosphate). To prevent possible multimerisation,oligomers designated BB2011 and BB2020 were not kinased. The sequencesof the oligomers shown in FIGS. 1 and 7 correspond to those given inSEQ. ID NOS:50-61.

The ligation products were separated on a 2% low gelling temperatureagarose gel and the DNA fragment of ca. 223 base pairs corresponding tothe hirudin HV-1 gene was excised and extracted from the gel. Thepurified fragment was then ligated to HindIII and EcoRI treated pUC19plasmid DNA. The transformation of E. coli host strains was accomplishedusing standard procedures. The strain used as a recipient in thetransformation of plasmid vectors was HW87 which has the followinggenotype:

araD139 (ara-leu) DELTA7697 (lacIPOZY) DELTA74 galU

galK hsdR rpsL srl recA56

The use of HW87 was not critical: any suitable recipient strain could beused, for example, E. coli AG1, which is available from NorthumbriaBiologicals Ltd. The recombinant ligation products were transformed intoE. coli K12 host strain HW87 and plated onto Luria-agar ampicillin (100μg/ml) plates. Twelve ampicillin-resistant colonies were picked and usedto prepare plasmid DNA for sequence analysis. Double stranded dideoxysequence analysis using a universal sequencing primer BB22(5'-CAGGGTTTTCCCAGTCACG-3'), (SEQ ID NO:3) complementary to theuniversal primer region of pUC19 was used to identify a correct clonepUC19 HV-1. The pUC19 recombinant was used to construct an expressionvector.

PREPARATION 2 Construction of a Hirudin HV1 Expression Vector

An expression vector was designed to enable the secretion of hirudin tothe extracellular medium after expression in S. cerevisiae. Secretion ofhirudin is desirable as this facilitates production of the protein withan authentic N-terminus. It also eases purification, limitsintracellular proteolysis, reduces potential toxic effects on the yeasthost and allows optimal protein folding and formation of nativedisulphide bonds. Secretion of hirudin through the yeast membrane wasdirected by fusion of hirudin to the yeast mating type alpha-factorpre-pro-peptide (a naturally secreted yeast peptide).

The yeast expression vector pSW6 (FIG. 2) is based on the 2μ circle fromS. cerevisiae. (pSW6 was deposited in S. cerevisiae strain BJ2168 at TheNational Collection of Industrial and Marine Bacteria Limited, 23 St.Machar Drive, Aberdeen, AB2 1RY, Scotland, United Kingdom on 23rd Oct.1990 under Accession No. NCIMB 40326. ) pSW6 is a shuttle vector capableof replication in both E. coli and S. cerevisiae and contains an originof DNA replication for both organisms, the leu2 gene (a selectablemarker for plasmid maintenance in the yeast host ) and the ampicillinresistant locus for selection of plasmid maintenance in E. coli. (TheDNA sequence of the vector has been determined; the E. coli sequencesare derived from the E. coli ColE1-based replicon pAT153. ) The fullsequence is given as SEQ.ID:4. The ability to passage this vectorthrough E. coli greatly facilitates its genetic manipulation and ease ofpurification. pSW6 contains an α-factor pre-pro-peptide gene fusedin-frame to the gene for epidermal growth factor (EGF). The expressionof this fusion is under the control of an efficient galactose regulatedpromoter which contains hybrid DNA sequences from the S. cerevisiae GAL1-10 promoter and the S. cerevisiae phosphoglycerate kinase (PGK)promoter. Transcription of the EGF gene is terminated in this vector bythe natural yeast PGK terminator. The EGF gene in pSW6 can be removed bydigestion with restriction endonucleases HindIII and BamHI. This removesDNA encoding both EGF and 5 amino acids from the C-terminus of theα-factor pro-peptide. Genes to be inserted into the pSW6 expressionvector must therefore have the general composition: HindIII siteα-factor adaptor - gene- BamHI site.

To rebuild the DNA encoding the amino acids at the C-terminal end of theα-factor pro-peptide and to fuse this to the synthetic hirudin gene, anoligonucleotide adapter (5'-AGCTTGGATAAAAGA-3' (top strand, SEQ. ID: 5), 5'-TCTTTTATCCA-3' (bottom strand, SEQ. ID: 6)) containing a HindIIIsite and codons encoding the Ser, Leu, Asp, Lys and Arg from theC-terminal end of the α-factor pro-peptide was constructed. The α-factoradaptor was ligated to the synthetic HV-1 gene such that the recombinantgene encoded an in-frame α-factor pro-peptide fusion to hirudin. ThepUC19 HV-1 plasmid DNA of Preparation 1 was first cleaved with BspMI andthe overhanging ends were filled using DNA polymerase I Klenow fragmentto create a blunt-ended linear DNA fragment. The linearised fragment wasseparated from uncut plasmid on a 1% low gelling temperature agarosegel, excised and extracted from the agarose gel matrix, then furthertreated with HindIII. The fragment was then ligated to the alpha-factoradaptor described above and annealed prior to ligation. The recombinantligation products were transformed into competent cells of E. colistrain HW87 (Preparation 1). Ampicillin resistant transformants wereanalysed by preparation of plasmid DNA, digestion with HindIII and BamHIand agarose gel electrophoresis. A correct recombinant plasmid wascalled pJC80. The α-factor adaptor hirudin sequence was removed frompJC80 on a ca. 223 bp HindIII-BamHI DNA fragment (SEQ.ID:7). Thefragment was purified on a low gelling temperature agarose gel andligated to HindIII and BamHI treated pSW6 vector DNA. The recombinantligation products were transformed into competent cells of E. colistrain HW87. Ampicillin resistant transformants were screened bypreparation of plasmid DNA, restriction endonuclease analysis withHindIII and BamHI and agarose gel electrophoresis. A clone with thecorrect electrophoretic pattern pJK1 (FIG. 3) was identified. Thisplasmid is the basic vector used for wild-type hirudin HV-1 expressionand was used to derive certain other yeast expression vectors asdetailed in the remaining preparations and examples.

PREPARATION 3 Expression of Hirudin Synthetic Gene

Plasmid expression vector pJK1 of Preparation 2 was transformed intoyeast (S. cerevisiae) strain BJ2168 which has the followinggenotype:prc-1-407, prb1-1122 pep4-3 leu2 trp1 ura3-52 cir+ using themethod of Sherman F. et al (Methods in Yeast Genetics, Cold SpringHarbor Laboratory, (1986)). All yeast media was as described by Shermanet al. Using 2 liter shake flasks, cultures of yeast containing pJK1were grown in 1 liter batches of 0.67% synthetic complete medium, yeastnitrogen base, with amino acids minus leucine and 1% glucose as a carbonsource. After overnight growth at 30° C., the cells were harvested bycentrifugation at 3000 rpm for 10 minutes and resuspended in the samesynthetic complete medium except that 1% galactose and 0.2% glucose wasused as the carbon source. This induces gene expression from the hybridPGK promoter. Cells were grown in the induction medium for 3 days. Afterthis period, the supernatant was harvested and assayed for hirudinactivity as described in Example 2, Section D, below.

EXAMPLE 1 Construction of a Hirudin-IEGR-Hirudin Fusion Gene and aVector for its Expression

A factor Xa-cleavable hirudin fusion protein molecule has beenengineered in which two full length hirudin molecules are joined by thepeptide linker sequence Ile Glu Gly Arg (See SEQ.ID: NOS:8 and 9). Themolecule is designed to be activatable by factor Xa cleavage. Thestrategy for construction of the hirudin-IEGR-hirudin gene is detailedbelow.

A gene encoding the hirudin-IEGR-hirudin molecule was constructed byoligonucleotide directed mutagenesis and molecular cloning. Mutagenesiswas carried out according to the method of Kunkel et al., Methods inEnzymology, 54, 367-382 (1987). Host strains are described below.

E. coli strains

RZ1032 is a derivative of E. coli that lacks two enzymes of DNAmetabolism: (a) dUTPase (dut), the lack of which results in a highconcentration of intracellular dUTP, and (b) uracil N-glycosylase (ung)which is responsible for removing mis-incorporated uracils from DNA(Kunkel et al., loc. cit.). A suitable alternative strain is CJ236,available from Bio-Rad Laboratories, Watford WD1 8RP, United Kingdom.The principal benefit is that these mutations lead to a higher frequencyof mutants in site directed mutagenesis. RZ1032 has the followinggenotype:

HfrKL16PO/45[lysA961-62), dut1, ung1, thi1,

recA, Zbd-279::Tn10, supE44

JM103 is a standard recipient strain for manipulations involving M13based vectors. The genotype of JM103 is DELTA (lac-pro), thi, supE,strA,endA, sbcB15, hsp4, F' traD36, proAB, lacIq, lacZDELTAM15. A suitablecommercially available alternative E. coli strain is E. coli JM109,available from Northumbria Biologicals Ltd.

Mutagenesis

Prior to mutagenesis it was neccesary to juxtapose two adjacent hirudingenes in an M13 mutagenesis vector. This was accomplished as describedbelow. pJK1 vector DNA of Preparation 2 was prepared and an aliquottreated with restriction endonucleases BglII and BamHI, a ca. 466 bpBglII-BamHI DNA fragment from this digestion was gel purified andligated to BamHI treated and phosphatased pJC80 vector DNA ofPreparation 2. The recombinant ligation products were transformed intocompetent cells of E. coli strain HW87 (Preparation 1). Ampicillin (100μg/ml) resistant clones were analysed by plasmid DNA preparation,restriction endonuclease digestion and gel electrophoresis. Clones withinserts in the desired orientation were identified after digestion withKpnI which released a DNA fragment of ca. 465 bp in length. (Theproducts of KpnI digestion were analysed on an agarose gel.) One of thecorrect clones, pJK002, was used for the remaining constructions, thisvector contains a ca. 465 bp KpnI DNA fragment which encodes aC-terminal portion of a first hirudin gene, a complete α-factorpre-pro-peptide sequence and the N-terminal portion of a second hirudingene. In order to delete the α-factor pre-pro-peptide sequence and toinsert DNA encoding a factor Xa-cleavable amino acid linker sequence(IEGR), the ca. 465 bp KpnI DNA fragment was transferred into abacteriophage mutagenesis vector M13mp18. Plasmid DNA of pJK 002 wasprepared and a portion was digested with KpnI. The ca. 465 bp KpnI DNAfragment from pJK002 was gel purified and ligated to KpnI treated andphosphatased M13mp18. The recombinant ligation products were transfectedinto competent cells of E. coli strain JM103. Single stranded DNAs fromputative recombinant phage plaques were prepared and analysed by dideoxysequence analysis using the M13 universal sequencing primer (SEQ. ID NO:11; see below). A clone pGC609 containing the KpnI fragment in thecorrect orientation was identified. The α-factor pre-pro-peptidesequence between the two hirudin sequences of pGC609 was deleted and theDNA encoding the Factor Xa-cleavable amino acid linker (IEGR) insertedby site directed mutagenesis. Single stranded DNA of pGC609 was preparedfrom E. coli strain RZ1032 and was used as a template for mutagenesiswith a 46mer oligonucleotide BB2988:(5'-CAGTCGGTGTAAACAACTCTTCCTTCGATCTGCAGATATTCTTCTG-3') (SEQ. ID: NO:10).Single stranded DNAs were prepared from putative mutant plaques and wereanalysed by dideoxy DNA sequence analysis using an M13 universalsequencing primer (United States Biochemical Corporation. P.O. Box22400, Cleveland, Ohio 44122. USA. Product No. 707635'-GTTTTCCCAGTCACGAC-3'), (SEQ. ID: NO:11). A correct clone, pGC610, wasidentified. To construct the full length hirudin-IEGR-hirudin gene thecentral core of the fusion molecule encoded on the ca. 210 bp KpnIfragment of pGC610 was cloned into the KpnI site of pJC80 of Preparation2. Replicative form DNA of pGC610 was prepared and digested with KpnI.The ca. 210 bp KpnI DNA fragment encoding the central core of thehirudin-IEGR-hirudin protein was gel purified and ligated to KpnItreated and phosphatased pJC80 of Preparation 2. The recombinantligation products were transformed into competent cells of E. colistrain HW87 (Preparation 1). Ampicillin (100 μg/ml) resistanttransformants were analysed by preparation of plasmid DNA, restrictionendonuclease digestion with PstI and agarose gel electrophoresis. Aclone with the correct electrophoretic pattern pDB1 was identified ascontaining a ca. 210 bp DNA fragment after PstI digestion.

To create a vector for the expression of the factor Xa-cleavablehirudin-IEGR-hirudin fusion protein the gene was cloned into the yeastexpression vector pSW6 of Preparation 2. Plasmid DNA of pDB1 was treatedwith HindIII and BamHI and the ca. 420 bp HindIII-BamHI DNA fragmentcontaining the factor Xa-cleavable hirudin-IEGR-hirudin gene was gelpurified and ligated to HindIII and BamHI treated pSW6 DNA ofPreparation 2. The recombinant ligation products were transformed intocompetent cells of E. coli strain HW87. Ampicillin (100 μg/ml) resistanttransformants were screened by preparation of plasmid DNA, restrictionendonuclease analysis with HindIII and BamHI and agarose gelelectrophoresis. A clone with the correct electrophoretic pattern pDB2was identified. pDB2 contained the hirudin-IEGR-hirudin gene fused inframe to the yeast α-factor pre-pro-peptide sequence. pDB2 plasmid DNAwas prepared and used to transform yeast strain BJ2168 (Preparation 3)according to the method of Sherman F. et al (Methods in Yeast Genetics,Cold Spring Harbor Laboratory, New York (1986)).

EXAMPLE 2 Purification of Hirudin and Hirudin-IEGR-Hirudin

The procedure of Preparation 3 was generally followed for the expressionof hirudin and hirudin-IEGR-hirudin proteins. Hirudin andhirudin-IEGR-hirudin are purified from yeast culture broth. Cells werefirst removed by centrifugation at 3000 rpm for 10 minutes. Thesupernatant was then assayed for biological activity using a chromogenicassay (see below, section D). Production levels from shake flaskcultures were routinely between 10-15 mg/liter of culture. The hirudinprotein was purified by preparative HPLC (DYNAMAX (Trade Mark) C18, 300angstroms). The column was first equilibrated in 15% acetonitrile, 0.1%trifluoro acetic acid. Then 2.5-3 mg of hirudin activity as determinedby chromogenic assay (section D) was loaded onto the column. The proteinwas eluted using a 15-40% acetonitrile gradient at 3 ml/minute over 25min. The purity of the isolated protein was assessed by analytical HPLC(VYDAC (Trade Mark) C18 reverse phase), N-terminal sequence analysis andmono Q FPLC as described below.

A. Assessing Purity by Analytical HPLC

Samples were analysed on a VYDAC (Trade Mark) C18 column (15×0.46 cm,particle size 5 micron) equilibrated with 10% acetonitrile, 0.1%trifluroacetic acid (TFA). Purified protein (20 μg) was loaded in 10%acetonitrile, 0.1% TFA. Protein was eluted at a flow rate of 1 ml/minuteusing an acetonitrile gradient from 10-40% in 0.1% TFA over 30 minutes.The eluted protein sample was monitored by absorbance at 280 nm.

B. Analysis of Purity by Mono Q FPLC

Samples were analysed on a Mono Q FPLC column (5×0.5 cm, Pharmacia)equilibrated in 20 mM Tris.HCl pH 7.5. Approximately 15 μg oflyophilised protein was reconstituted in 1 ml 20 mM Tris. HCl pH 7.5 andloaded onto the column. Protein was eluted using a gradient of 0-250 mMNaCl in 20 mM Tris. HCl buffer (pH 7.5) at a flow rate of 1 ml/minuteover 30 minutes.

C. N-terminal Sequence Analysis

N-terminal sequence analysis was performed by automated Edmandegradation using an Applied Biosystems Protein Sequencer, model 471 A(Applied Biosystems, Foster City, Calif.).

Purified material that was greater than 95% pure, was dried down in aSPEEDIVAC (trade mark of Savant Instruments Inc. Hicksville, N.Y.U.S.A.) and reconstituted in 0.5 ml of 0.9% (w/v) saline for assay.

D. Hirudin Anti-thrombin Chromogenic Activity Assay

The ability of hirudin and molecules containing hirudin to inhibit thethrombin catalysed hydrolysis of the chromogenic substratetosyl-Gly-Pro-Arg-p-nitroanilide (CHROMOZYM TH (trade mark ofBoehringer-Mannheim)) was used as an assay to determine theiranti-thrombin activity. Protein samples (50 μl ) diluted in 0.1MTris.HCl pH8.5, 0.15M NaCl, 0.1% (w/v) PEG 6000 were mixed with 50 μlhuman thrombin (Sigma, 0.8 U/ml in the above buffer) and 50 μl CHROMOZYMTH (2.5 mM in water) in 96 well plates (Costar). The plates wereincubated at room temperature for 30 minutes. The reaction wasterminated by adding 50 μl 0.5M acetic acid and the absorbance read at405 nm using an automatic plate reader (Dynatech). Quantitation wasperformed by comparison with a standard hirudin preparation (recombinant[Lys-47]-HV-2 purchased from Sigma: Sigma Chemical Co. Ltd, Fancy Road,Poole, Dorset BH11 7TG, United Kingdom).

EXAMPLE 3 Cleavage and Activation of Hirudin-IEGR-Hirudin Fusion Protein

Purified hirudin-IEGR-hirudin fusion protein was incubated with FactorXa. The reaction was performed at 37° C. in a total volume of 150 μl of0.1M Tris.HCl buffer pH 7.8 and contained 2.06 nmol fusion protein and0.4 nmol Factor Xa. Analysis of the reaction mixture by sodium dodecylsulphate-polyacrylamide gel electro- phoresis (SDS-PAGE) demonstratedcleavage to products of a similar size to native hirudin. Reverse phaseHPLC analysis of the cleavage reaction as in Example 2, section A,demonstrated the appearance of two new species with retention times (RT)of 17 and 20 minutes compared to 22 minutes for the intact fusionprotein.

Measurements of specific activity were made on the products isolatedfrom a cleavage reaction. Using a chromogenic assay according to themethod of Example 2, section D, to measure hirudin activity inanti-thrombin units and A 280 nm to determine protein concentration, thefollowing results were obtained: product RT 17 min., 6125 U/mg; productRT 20 min., 5226 U/mg; intact hirudin-IEGR-hirudin, RT 22 min., 588U/mg. Cleavage therefore produces an approximate 2-fold increase inspecific activity, with the products displaying similar values to thatrecorded for a recombinant hirudin sample (6600 U/mg) as measuredaccording to the method of Example 2, section D.

Purified cleavage products and the intact fusion protein were subjectedto N-terminal sequence analysis.

In each case the sequence obtained was identical to that of nativehirudin (HV-1), (VVYTD); SEQ ID NO: 70.

It has thus been demonstrated that the hirudin-IEGR-hirudin fusionprotein can be cleaved by Factor Xa to produce two products with hirudinactivated. Cleavage of the fusion protein is accompanied by activationas the products of cleavage have approximately double the specificactivity of the fusion protein.

PREPARATION 4 Isolation of a Streptokinase Gene

Streptokinase is secreted by Lancefield's Group C streptococci andcloning of the streptokinase gene from Streptococcus equisimilis strainH46A has been described (Malke, H. and J. J. Ferretti, P.N.A.S. 813557-3561 (1984)). The nucleotide sequence of the cloned gene has beendetermined (Malke, H., Roe, B. and J. J. Ferretti, Gene 34 357-362(1985)). A gene encoding streptokinase has been cloned from S.equisimulis (ATCC 9542 or ATCC 10009) for use in the current invention.Methods that can be used to isolate genes are well documented and theprocedure used to isolate the streptokinase gene is summarized in thefollowing protocol.

1. DNA was prepared either from Streptococcus equisimilis is(Lancefield's Group C) ATCC 10009 or from ATCC 9542 grown in brain-heartinfusion medium (Difco-Bacto Laboratories, PO Box 14B, Central Avenue,E. Mosely, Surrey KT8 OSE, England) as standing cultures. ChromosomalDNA was isolated from approximately 1.5 ml of cells at a density of1×10¹¹ cells/ml. The cells were harvested and washed in 1 ml buffer(0.1M potassium phosphate pH 6.2). The pellet was resuspended in 400 μlof the same buffer and 500 units of mutanolysin (Sigma Chemical CompanyLtd, Fancy Road, Poole, Dorset BH17 7TG, UK) in 100 μl volume was added.This mix was incubated at 37° C. for 1 hour. The cells were harvested bycentrifugation and again washed in buffer. The cells were resuspended in500 μl of a solution containing 50 mM glucose, 10 mM EDTA and 25 mM TrisHCl pH 8.0 and incubated at 37° C. for approximately 1 hour with the mixbeing shaken gently to prevent the cells settling. A 500 μl aliquot of asolution containing 0.4% SDS and proteinase K (100 μg/ml) (SigmaChemical Company Ltd) was added and the mix was incubated at 37° C. for1 hour until it became viscous and clear. The mix was then extractedthree times with phenol equilibrated with TE buffer (10 mM Tris HCl 1 mMEDTA pH 8.0). The aqueous phase was removed into an eppendorf tube,sodium acetate added to a final concentration of 0.3M and 2.5 volumes ofethanol added. The mix was incubated at -70° C. for 1 hour toprecipitate the DNA. The DNA was pelleted by centrifugation, washed with70% ethanol and then resuspended in 200 μl TE buffer.

2. The Polymerase Chain Reaction (PCR) was used to amplify thestreptokinase sequence (Saiki R. et al Science, 239, 487-491 (1988)).Two primers were designed based on the published streptokinasesequences. The primer encoding the antisense strand at the 3' end of thegene was a 40mer BB1888 (5'GTTCATGGATCCTTATTTGTCGTTAGGGTTATCAGGTATA 3'), (SEQ. ID: NO:12) which also encoded a BamHI site. The primer encodingthe sense strand at the 5' end of the gene encoded an EcoRI site inaddition to the streptokinase sequence and was the 40 mer BB1887(5'TCAAGTGAATTCATGAAAAATTACTTATCTTTTGGGATGT 3'), (SEQ ID: NO:13). Fortycycles of PCR were performed with the denaturation step at 95° C. for 2minutes, followed by annealing of the primers for 3 minutes at 55° C.and extension at 70° C. for 4.5 minutes. A sample of the reactionproduct was analysed on a 0.8% agarose gel. A single amplified DNAfragment at c.a. 1.3 kB, which corresponds to the expected size of thestreptokinase gene, was observed.

3. A 30 μl sample of the product was digested with the restrictionendonucleases EcoRI and BamHI, analysed on a low gelling temperatureagarose gel and the c.a. 1.3 kb DNA fragment was isolated from the gel.The band was extracted from the gel and ligated into the plasmid pUC19which had been cleaved with EcoRI and BamHI to form the plasmid pUC19SK.

The entire ca. 1330 bp EcoRI-BamHI fragment from pUC19SK was sequencedby dideoxy sequence analysis. To facilitate the sequencing, TheEcoRI-BamHI DNA fragment of pUC19SK was transferred to M13 sequencingvectors mp18 and mp19 in two halves. A ca. 830 bp EcoRI-HindIII DNAfragment was separately transferred into EcoRI and HindIII treatedM13mp18 and M13mp19. The products from these two ligation events wereseparately transfected into competent cells of E. coli host JM103.Single stranded DNA was prepared and used for dideoxy sequence analysisusing the primers listed in SEQ ID NOS: 62 to 68 and SEQ ID NO: 11. Aca. 490 bp HindIII-BamHI fragment was gel purified after treatment ofpUC19SK with HindIII and BamHI. This DNA fragment was separately ligatedto M13mp18 and M13mp19 which had been treated with HindIII and BamHI.The products of these two ligations was transfected into competent cellsof E. coli host JM103. Single stranded DNA was prepared and used fordideoxy sequence analysis with the primers shown in SEQ ID NOS:62 to 68and SEQ ID NO: 11. The entire sequence of the EcoRI-BamHI PCR derivedDNA fragment is shown in SEQ ID NOS:14. and 15.

EXAMPLE 4 Construction of Streptokinase Expression Vectors

A number of alternative streptokinase expression vectors have beenconstructed for expression in either yeast S. cerevisiae or E. coli K12.

1) Vectors for secretion to the periplasm of E. coli K12

Two vectors were designed to enable the secretion of streptokinase tothe periplasmic space after expression in E. coli K12. Secretion ofstreptokinase is desirable to facilitate production of protein with anauthentic N-terminus, to ease purification, to reduce potential toxiceffects and to limit intracellular proteolysis. Secretion ofstreptokinase through the E. coli cytoplasmic cell membrane was directedby either the streptokinase signal peptide or the E. coli major outermembrane protein A (OmpA) signal peptide (OmpAL).

A. Secretion using the streptokinase leader

The streptokinase gene of Preparation 4 was transferred into the E. coliexpression vector pGC517 (FIG. 4). pGC517 contains the regulatable ptacpromoter, a ribosome binding site and a synthetic transcriptionalterminator. pGC517 was deposited in E coli K12 at The NationalCollection of Industrial and Marine Bacteria Limited, 23 St. MacharDrive, Aberdeen, AB2 1RY, Scotland, United Kingdom on 5th December 1990under Accession No. NCIMB 40343. Genes can be cloned into the expressionsite of pGC517 on NdeI-BamHI DNA fragments. It was necessary to engineera NdeI site into the 5' end of the streptokinase gene to enablesubsequent cloning into pGC517. The NdeI site was introduced bysite-directed mutagenesis. To construct the vector for the site directedmutagenesis, plasmid DNA of vector pUC19SK of Preparation 4 was preparedand digested with EcoRI and BamHI and the ca. 1.3 Kb EcoRI-BamHI DNAfragment was gel purified and ligated to M13mp18 treated with EcoRI andBamHI. Recombinant ligation products were transfected into competentcells of E. coli strain JM103 (Example 1). Single stranded DNA wasprepared from the putative recombinant plaques and analysed by dideoxysequence analysis using the M13 universal sequencing primer (SEQ ID: NO:11 of Example 1). One of the correct recombinant phages was calledpGC611. Single stranded DNA of phage pGC611 was prepared from E. colistrain RZ1032 (Example 1) and used as a template for mutagenesis. AnNdeI restriction site was introduced by site-directed mutagenesis at the5' end of the streptokinase gene such that the NdeI site overlapped thestreptokinase initiation codon. The mutagenesis was performed using a26-mer BB2175 (5'-GATAAGTAATTTTTCATATGAATTCG-3'), (SEQ ID: NO:16).Single stranded DNAs were prepared from putative mutant plaques and werescreened by dideoxy sequence analysis using the 18mer sequencing primerBB2358 (5'-CATGAGCAGGTCGTGATG-3'), (SEQ ID: NO:17) and a correct clonepGC612 was identified.

To construct an expression vector, the streptokinase gene carrying thenewly introduced NdeI site, was cloned into the pGC517 expressionvector. Replicative form DNA was prepared from pGC612 and was digestedwith NdeI and BamHI and the ca. 1.3 kb NdeI-BamHI DNA fragment was gelpurified. This fragment was then ligated to NdeI and BamHI treatedpGC517 DNA. The recombinant ligation products were transformed intocompetent cells of E. coli strain JM103. Ampicillin (100 μg/ml)resistant transformants were analysed by plasmid DNA preparation,restriction endonuclease digestion with BglII and BamHI and agarose gelelectrophoresis. One of the correct clones, pKJ2, was verified bydideoxy sequence analysis using the sequencing primer BB2358. Thisvector contains the entire streptokinase gene including the sequencesencoding the streptokinase signal peptide leader region and was used forthe expression of streptokinase in E. coli.

B. Secretion using the E. coli OmpA leader

As an alternative secretion signal, a DNA sequence encoding the majorouter membrane protein A (OmpA) signal peptide (OmpAL) was fused to theDNA sequence encoding the mature streptokinase protein; see SEQ ID NOS:18 and 19 A DNA fragment encoding streptokinase was obtained bypreparing pUC19SK vector DNA, treating the DNA with EcoRI and filling-inthe overhanging single stranded DNA ends with DNA polymerase I Klenowfragment to create a blunt-ended linear DNA fragment. The fragment wasnext digested with BamHI and the ca. 1.3 kb blunt-ended-BamHI DNAfragment containing the streptokinase gene was gel-purified. The DNAsequence encoding OmpAL is available on an expression vector pSD15. ThepSD15 vector contains a gene encoding an insulin like growth factor IIgene (IGF-II) fused to the OmpAL signal sequence. pSD15 was deposited inE. coli K12 at The National Collection of Industrial and Marine BacteriaLimited, 23 St. Machar Drive, Aberdeen, AB2 1RY, Scotland, UnitedKingdom on 5th December 1990 under Accession No. NCIMB 40342. In orderto use pSD15 as a vector to provide the OmpAL DNA sequence, pSD15 vectorDNA was treated with NheI, the single stranded DNA overhanging ends werefilled-in with DNA polymerase I Klenow fragment to create a blunt-endedlinear DNA fragment. The linear DNA fragment was next digested withBamHI which removed ca. 123 bp from the 3' end of the IGF-II gene inpSD15. After restriction endonuclease digestion the cleaved linear DNAfragment was treated with phosphatase, to prevent recircularisation ofany partially cut vector DNA and was gel purified then ligated to theblunt-ended-BamHI DNA fragment containing the streptokinase gene. Theligated mixture was transformed into competent cells of E. coli strainHW87 (Preparation 1). Ampicillin (100 μg/ml) resistant recombinantscarrying the streptokinase gene were characterised by preparation ofplasmid DNA, restriction endonuclease analysis with BglII and HindIIIand agarose gel electropohoresis. A construct of the correctelectrophoretic pattern was called pKJ1. Vector pKJ1 contains the DNAencoding OmpAL and streptokinase separated by a region of DNA notrequired in further constructs. The sequence of the insert DNA in pKJ1was confirmed by dideoxy sequence analysis with a 44-mer oligonucleotideBB58 (5'-AGCTCGTAGACACTCTGCAGTTCGTTTGTGGTGACCGTGGCTTC-3') SEQ ID: NO:20.In order to create a DNA template for the deletion loopout mutagenesisof the unwanted DNA sequence, the BglII to HindIII DNA fragment frompKJ1 was cloned into a vector M13mp19. pKJ1 vector DNA was treated withBglII and HindIII to produce a ca. 1026 bp DNA fragment, which was gelpurified and ligated into the polylinker region of M13mp19 replicativeform DNA treated with BamHI and HindIII. Ligation products weretransfected into competent cells of E. coli strain JM103. Singlestranded DNAs were prepared from putative recombinant plaques and acorrect clone (pGC600) identified by dideoxy sequence analysis using theM13 universal sequencing primer (SEQ ID: NO:11, Example 1).

Mutagenesis on template pGC600 was performed using a 30-meroligonucleotide mutagenesis primer BB2658(5'-ACCGTAGCGCAGGCCATTGCTGGACCTGAG-3') SEQ ID: NO: 21. Single strandedDNAs were prepared from putative mutant plaques and a clone, pGC601,containing the required deletion was identified using dideoxy sequenceanalysis with the M13 universal sequencing primer (SEQ ID: NO: 11).pGC601 contains part of the OmpAL-streptokinase fusion required for thesecretion of streptokinase from this signal peptide in E. coli, but DNAencoding the C-terminal portion of streptokinase is absent. In order toreconstruct the streptokinase gene, replicative form DNA from pGC601 wasdigested with restriction enzymes NdeI and HindIII and the ca. 810 bpNdeI-HindIII DNA fragment containing the DNA sequences encoding OmpALleader peptide sequence fused to the N-terminal portion of streptokinasewas gel purified. pJK2 vector DNA was treated with restriction enzymesNdeI and HindIII followed by treatment with phosphatase and the ca. 3620bp NdeI-HindIII vector DNA fragment containing the essential vectorsequences and the C-terminal portion of the streptokinase gene was gelpurified. The ca. 810 bp NdeI-HindIII (pGC601) and ca. 3620 NdeI-HindIII(pKJ2) gel purified DNA fragments were ligated together and therecombinant ligation products were transformed into competent cells ofE. coli strain HW1110 (lacIq). The lacIq mutationin this strain enhancesrepression of transcription from the tac promoter. Any other lacIqstrain, for example JM103 could be used instead. The ampicillinresistant transformants were screened by preparation of plasmid DNAfollowed by restriction endonuclease analysis using NdeI and HindIII.Agarose gel electrophoresis of digestion products was used to identify acorrect clone which was called pLGC1. The pLGC1 construct was verifiedby dideoxy sequence analysis using a 17-mer oligonucleotide BB2753(5'-GACACCAACCGTATCAT-3'), (SEQ ID: NO: 22) to sequence through theBamHI site and primer BB3510 (5'-CACTATCAGTAGCAAAT-3'), (SEQ ID: NO:23)to sequence through the sequence encoding the OmpA leader.

2) Intracellular Expression in E. coli

As streptokinase contains no disulphide bonds there is no requirementfor secretion to encourage native protein folding and althoughstreptokinase is naturally secreted, intracellular expression offersseveral potential advantages such as high yield and inclusion bodyformation which may facilitate purification. As an alternativeproduction route, an expression vector was designed for intracellularproduction of streptokinase in E. coli. DNA encoding the amino acids 2to 21 of the OmpAL signal peptide sequence which was fused to maturestreptokinase in pGC601 were deleted by loopout site directedmutagenesis using single stranded DNA of pGC601 with a 31-mermutagenesis oligonucleotide BB3802(5'-GAAATACTTACATATGATTGCTGGACCTGAG-3'), (SEQ ID: NO:22). In addition todeleting the OmpAL signal peptide coding sequence, BB3802 fused themethionine codon (ATG) of the OmpAL signal peptide sequence to the firstcodon of mature streptokinase to create the 5' end of gene encoding aMethionyl-streptokinase fusion protein (see SEQ ID: NOS: 25 and 26). TheATG codon was used to allow initiation of translation at the correctposition. Single stranded DNA was prepared from putative mutant plaquesand a clone containing the desired mutation, pGC602 was identified usingdideoxy sequence analysis with the M13 universal sequencing primer (SEQID: NO: 11). The C-terminal portion of the streptokinase gene is missingin pGC602. In order to reconstruct the intact mature streptokinasecoding sequence, replicative form DNA from pGC602 was digested withrestriction enzymes NdeI and HindIII and the ca. 755 bp NdeI-HindIII DNAfragment encoding the N-terminal portion of the Methionyl-streptokinaseprotein was gel purified and ligated to the gel purified ca. 3620 bpNdeI-HindIII pLGC2 vector DNA fragment described in Example 6 below. Therecombinant ligation mixture was transformed into competent cells of E.coli strain HW1110 (lacIq). Ampicillin (100 μg/ml) resistanttransformants were screened by plasmid DNA preparation, restrictionendonuclease digestion and agarose gel electrophoresis. A clone, pGC603,with the correct electrophoretic pattern after NdeI and HindIIIdigestion, was identified. Vector pGC603 was used for the intracellularexpression of Methionyl-streptokinase in E. coli strain HW1110.

3) Construction of Expression Vectors for the Secretion of Streptokinasefrom the Yeast S. cerevisiae

Expression vectors were designed to enable the secretion ofstreptokinase to the extracellular medium after expression in S.cerevisiae. Secretion of streptokinase is desirable to facilitateproduction of protein with an authentic N-terminus, to easepurification, to limit intracellular proteolysis and to reduce potentialtoxic effects on the yeast host. Secretion of streptokinase through theyeast membrane was directed by either the natural streptokinase signalpeptide or by fusion of mature streptokinase to the yeast mating typealpha-factor pre-pro-peptide (a naturally secreted yeast peptide) seeSEQ ID: NOS:27 and 28.

A) Secretion of Streptokinase using the Streptokinase Signal Peptide

The streptokinase gene with its natural signal peptide was cloned intothe yeast expression vector pSW6 to allow its expression in the yeast S.cerevisiae. Vector DNAs of pKJ2 and pSW6 of Preparation 2 were prepared.Both DNAs were treated with restriction enzymes BglII and BamHI and theca. 1420 bp DNA fragment from pKJ2 and the ca. 7460 bp vector DNAfragment from pSW6 were gel purified and ligated together. Therecombinant ligation products were transformed into competent cells ofE. coli strain DH5 (supE44, hsdR17, recA1, endA1, gyrA96, thi-1, relA1),but any other good transforming strain could be used, for example JM109of Example 1. Ampicillin (100 μg/ml) resistant transformants wereanalysed by preparation of plasmid DNA, restriction endonucleasedigestion with BamHI and HindIII and agarose gel electrophoresis. Aclone with the correct electrophoretic pattern pSMD1/111 was used forthe expression of streptokinase from its own signal peptide sequencefrom the yeast S. cerevisiae. Plasmid expression vector pSMD1/111 wastransferred into yeast (S. cerevisiae) strain BJ2168 according to themethod of Preparation 3.

B) Secretion of Streptokinase using the pre-pro-α-Factor SecretionLeader

A gene fusion to enable the streptokinase gene of Preparation 4 to beexpressed in yeast and to be secreted by the yeast mating type α-factorpre-pro-peptide was designed and constructed using site-directedmutagenesis and molecular cloning see SEQ ID: NOS:27 and 28. Theconstruction involved mutagenesis to create an α-factor-streptokinasefusion gene and molecular cloning to reconstruct the DNA sequencesencoding the mature streptokinase protein sequence. Single stranded DNAof pGC600 prepared from E. coli strain RZ1032 (Example 1) was used as amutagenesis template with the 36-mer oligonucleotide BB3624(5'-GTCCAAGCTAAGCTTGGATAAAAGAATTGCTGGACC-3') SEQ ID: NO:29. Singlestranded DNA from putative mutant plaques were analysed by dideoxysequence analysis using the M13 universal sequencing primer (SEQ ID:NO:11) and a mutant clone, pGC614, with the desired sequence wasidentified. In pGC614 the OmpA-IGFII-Streptokinase signal peptideencoding sequences of pGC600 have been deleted and the α-factor linkerencoding the C-terminal 5 amino acids of the α-factor pro-peptidedescribed in Preparation 2 have been inserted. To reconstruct thestreptokinase gene in a yeast expression vector, two stages of geneticmanipulation were required. First the C-terminal portion ofstreptokinase was cloned into a yeast expression vector and this newconstruct was used to clone in the N-terminal α-factor-streptokinasefusion portion of the gene, thus reconstructing a mature streptokinasecoding region fused to the α-factor pre-propeptide gene. Vector DNAs ofpKJ2 and pSW6 (Preparation 2) were prepared and digested with HindIIIand BamHI and the ca. 485 bp. DNA fragment from pKJ2 and the ca. 7750bp. vector DNA fragment from pSW6 were gel purified and ligated.Recombinant ligation products were transformed into competent cells ofE. coli strain DH5. Ampicillin resistant transformants were screened bypreparation of plasmid DNA, restriction endonuclease digestion withHindIII and BamHI and agarose gel electrophoresis. A clone with thecorrect electrophoretic pattern pSMD1/119 was isolated. It contains DNAencoding the C-terminal portion of streptokinase cloned into a yeastexpression vector. The DNA encoding the N-terminal portion ofstreptokinase and the alpha-factor adaptor sequence were next clonedinto pSMD1/119. Replicative form DNA of pGC614 was prepared and treatedwith HindIII and ligated to pSMD1/119 vector DNA which had been treatedwith HindIII and phosphatased. The recombinant ligation products weretransformed into competent cells of E. coli strain DH5. Ampicillin (100μg/ml) resistant transformants were screened by preparation of plasmidDNA, restriction endonuclease analysis with DraI and agarose gelelectrophoresis. A clone with the correct electrophoretic patternpSMD1/152 gave DraI digestion products of ca. 4750, 1940, 1520 and 700bp. in length. pSMD1/152 was used for the expression and secretion ofstreptokinase using the alpha factor pre-pro-sequence from the yeast S.cerevisiae. Plasmid expression vector pSMD1/152 was transferred intoyeast (S. cerevisiae) strain BJ2168 according to the method ofPreparation 3.

EXAMPLE 5 Construction of a Gene Encoding a Core Streptokinase Protein

A gene encoding a truncated methionyl streptokinase molecule (aa 16-383)was designed and constructed by oligonucleotide directed loopoutdeletions and molecular cloning; see SEQ ID: NOS:30 and 31 DNA encodingthe amino acids 2 to 21 of the OmpAL signal sequence, the DNA encodingIGF-II, the DNA encoding the streptokinase signal peptide and the first15 amino acids of the mature streptokinase protein in pGC600 of Example4B were deleted by loopout mutagenesis using a 33-mer oligonucleotideBB3862: 5'-GAAATACTTACATATGAGCCAATTAGTTGTTAG-3'; SEQ ID: NO:32. Singlestranded DNA was prepared from E. coli RZ1032 cells infected with pGC600and used as the template for mutagenesis with primer BB3862. Singlestranded DNA was prepared from putative mutant plaques and a clonepGC604 containing the desired deletion was identified by dideoxysequence analysis using the M13 universal sequencing primer (SEQ ID:NO:10, Example 1).

Amino acids 384 to 414 were deleted from streptokinase by loopoutmutagenesis using a 28-mer oligonucleotide BB3904:5'-CCCGGGGATCCTTAGGCTAAATGATAGC-3'; SEQ ID: NO: 33. The template for themutagenesis was single stranded DNA of M13JK1 of Example 10 containingthe ca. 500 bp HindIII-BamHI DNA fragment encoding the 3' end of thestreptokinase gene from pUC19SK of Preparation 4. Single stranded DNAfrom putative mutant plaques was prepared and a clone pGC605 containingthe desired deletion was identified by dideoxy sequence analysis usingthe M13 universal sequencing primer (SEQ ID: NO:11, Example 1).

The intact core streptokinase molecule was reconstructed from the twomutated halves by a two step ligation incorporating the NdeI-HindIII DNAfragment from pGC604 (containing the DNA encoding the N-terminal portionof the core streptokinase molecule) and the HindIII-BamHI DNA fragmentfrom pGC605 (containing the DNA encoding the C-terminal portion of thecore streptokinase molecule) into the vector DNA pLGC2 of Example 6below. First the pGC604 DNA was digested with NdeI and HindIII. A DNAfragment of ca. 710 bp. was gel purified. Vector DNA was prepared frompLGC2 of Example 6 and treated with NdeI and HindIII and phosphatased.The linear vector DNA was gel purified and the two fragments wereligated together. The recombinant ligation products were transformedinto competent cells of E. coli strain HW1110. Ampicillin (100 μg/ml )resistant transformants were screened for the required clone bypreparation of plasmid DNA, restriction endonuclease analysis with NdeIand HindIII followed by agarose gel electrophoresis of the digestionproducts. One construct with the correct electrophoretic pattern,pGC617, was identified.

To clone the DNA encoding the C-terminal portion, the same vector DNA(pLGC2) was treated with HindIII and BamHI and phosphatased. The pGC605DNA was treated with HindIII and BamHI and a ca. 402 bp DNA fragment wasgel purified and ligated into the HindIII and BamHI treated pLGC2 vectorDNA. The recombinant ligation products were transformed into competentcells of E. coli strain HW1110. Ampicillin (100 μg/ml) resistanttransformants were screened for the required clone by preparation ofplasmid DNA, restriction endonuclease analysis with BamHI and HindIII,and agarose gel electrophoresis of the digestion products. One constructwith the correct electrophoretic pattern pGC618 was identified. Finally,to reconstruct the intact core streptokinase gene from the two halves,pGC617 DNA was treated with HindIII and BamHI and the ca. 402 bpHindIII-BamHI fragment from pGC618 ligated to it. pGC618 DNA wasdigested with HindIII and BamHI and a ca. 402 bp HindIII-BamHI DNAfragment was gel purified. pGC617 vector DNA was also treated withHindIII and BamHI and a ca. 402 bp HindIII-BamHI DNA fragment frompGC618 was ligated into it. The ligation products were transformed intocompetent cells of E. coli strain HW1110. Ampicillin resistanttransformants were screened by preparation of plasmid DNA restrictionendonuclease analysis with BamHI and HindIII and agarose gelelectrophoresis. A correct construct, pGC606, was identified.

EXAMPLE 6 Construction of Expression vectors containing a ThrombinCleavable Streptokinase-Streptokinase Fusion Gene 1) Construction of aSecretion Vector for the Expression of a Thrombin CleavableStreptokinase-Streptokinase Fusion

A gene encoding an OmpAL streptokinase-streptokinase fusion linked by athrombin cleavable linker sequence VELQGVVPRG, identical to that at thethrombin cleavage site in Factor XIII, was designed and constructed bysite directed mutagenesis and molecular cloning (SEQ ID: NOS:34 and 35).A ca. 1.3 Kb EcoRI-BamHI DNA fragment containing a streptokinase genewas gel purified after treatment of the pUC19SK vector DNA ofPreparation 4 with EcoRI and BamHI. A second DNA fragment encoding astreptokinase gene was gel purified after BglII and SalI digestion ofthe pKJ1 vector DNA of Example 4. A trimolecular ligation was carriedout between these two fragments and EcoRI and SalI treated pGC517 vectorDNA described in Example 4, section 1A. The recombinant ligationproducts were transformed into competent cells of E. coli strain HW1110(laqIq). Ampicillin (100 μg/ml) resistant transformants were screened bypreparation of plasmid DNA, restriction endonuclease analysis with EcoRIand SalI and agarose gel electrophoresis. A clone with the correctelectrophoretic pattern (pSD93) was identifed. pSD93 contains two tandemcopies of the streptokinase gene separated by a sequence containing thebacteriophage lambda gene cII ribosome binding site, and encoding theOmpA signal peptide sequence, the streptokinase signal peptide sequenceand the 5' part of the IGF-II sequence from pKJ1. To remove thisunwanted intervening sequence and to replace it with the desiredthrombin cleavable linker sequence a part of pSD93 was transferred intoan M13 mutagenesis vector for mutagenesis. Plasmid pSD93 DNA wasdigested with HindIII and a ca. 1530 bp DNA fragment gel purified andligated to HindIII treated and phosphatased replicative form M13mp18DNA. The recombinant ligation products were transformed into competentcells of E. coli strain JM103 (Example 1). There are two possiblefragment orientations in such a construction. The orientation of theclones was determined by preparation of replicative form DNA andanalysing the DNA fragments produced after XmnI digestion and agarosegel electrophoresis. One of the clones pSD95 which contained thefragment in an inverted orienation (thus preventing translationreadthrough by virtue of fusion to the α-fragment of β-galactosidaseexpressed from the M13 mutagenesis vector) was used for mutagenesis.Single stranded DNA template was prepared from pSD95 and used for sitedirected mutagenesis. The primer used was a 63-mer oligonucleotideBB2938:(5'-GATAACCCTAACGACAAAGTAGAGCTGCAGGGAGTAGTTCCTCGTGGAATTGCTGGACCTGAG-3')(SEQ ID: NO:36) designed to loop out the gene cII ribosome binding site,the OmpAL IGF-II sequence, the streptokinase signal peptide sequence inpSD95 and to insert a DNA sequence encoding a thrombin cleavable aminoacid sequence. Single stranded DNAs were prepared from putative mutantplaques and a correct mutant pGC607 was identified using dideoxysequence analysis with primer BB2753 (SEQ ID: NO:22) of Example 4.Replicative form DNA of pGC607 was prepared and was digested withHindIII and the ca. 1277 bp HindIII DNA fragment gel purified andligated to HindIII treated and phosphatased pLGC1 vector DNA of Example4. The recombinant ligation products were transformed into competentcells of E. coli strain HW1110. Ampicillin resistant transformants werescreened by preparation of plasmid DNA, restriction endonucleaseanalysis using HindIII and agarose gel electrophoresis. This cloningrebuilds the gene encoding a thrombin cleavablestreptokinase-streptokinase fusion in an expression vector. A clone(pLGC2) carrying the insert in the sense orientation was identified bydideoxy sequence analysis using primers BB2754 (5'-GCTATCGGTGACACCAT-3')SEQ ID: NO:37 and BB3639 (5'-GCTGCAGGGAGTAGTTC-3') SEQ ID: NO:38. pLGC2was used for the expression of thrombin cleavablestreptokinase-streptokinase fusion protein in E. coli HW1110.

2) Construction of a Vector for the Intracellular Expression of aThrombin Cleavable Streptokinase-Streptokinase Fusion Gene.

A thrombin cleavable methionyl -streptokinase-streptokinase gene wasdesigned and constructed by molecular cloning. The gene was constructedfrom the methionyl-streptokinase gene of Example 4 and the HindIII DNAfragment from pGC607 of Example 6, encoding the C-terminal portion of afirst streptokinase molecule, a thrombin cleavable linker and anN-terminal portion of a second streptokinase molecule.

Replicative form DNA of pGC607 was prepared and was digested withHindIII and the ca. 1277 bp HindIII DNA fragment was gel purified andligated to HindIII treated and phosphatased pGC603 vector DNA of Example4. The recombinant ligation products were transformed into competentcells of E. coli strain HW1110 (lacIq). Ampicillin (100 μg/ml) resistanttransformants were screened by preparation of plasmid DNA, restrictionendonuclease analysis with HindIII, BamHI and PstI and agarose gelelectrophoresis of the digestion products. One construct with thecorrect electrophoretic pattern pLGC3, was used for the intracellularexpression of a thrombin cleavable methionyl-streptokinase-streptokinasefusion protein.

EXAMPLE 7 Construction of a Thrombin Cleavable Core Streptokinase-coreStreptokinase Fusion Gene

A gene encoding a core methionyl -streptokinase-core streptokinasefusion linked by a thrombin cleavable linker sequence VELQGVVPRG (SEQID: NO: 69), identical to that at the thrombin cleavage site in FactorXIII, was designed and constructed by site directed mutagenesis andmolecular cloning see SEQ ID: NOS:39 and 40 The core streptokinase-corestreptokinase fusion gene was constructed from the core streptokinasemonomer gene of Example 5 and a HindIII DNA fragment containing theC-terminal portion of a core streptokinase gene, a thrombin-cleavablelinker and an N-terminal portion of a core streptokinase gene. Toconstruct the HindIII DNA fragment containing the appropriate deletionsand encoding a thrombin-cleavable linker, pGC607 of Example 6 was usedas a template for oligonucleotide directed mutagenesis. A 61-meroligonucleotide BB3861:(5'-GCTATCATTTAGCCGTAGAGCTGCAGGGAGTAGTTCCTCGTGGAAGCCAATTAGTTGTTAG-3')SEQ ID: NO:41 was used to delete the streptokinase amino acids 384 to414, to reconstruct the thrombin cleavable linker sequence VELQGVVPRGand to delete the first 15 amino acids of the N-terminus ofstreptokinase. Single stranded DNA from putative mutant plaques wasprepared and a correct clone, pGC608, was identified by dideoxy sequenceanalysis using sequencing primer BB2753 of example 8. Replicative formDNA was prepared from pGC608 and used in further construction.

To construct an intact core methionyl-streptokinase-core-streptokinasefusion, pGC608 DNA was treated with HindIII and the ca. 1140 bp HindIIIDNA fragment encoding the C-terminal portion of the core streptokinasemolecule, the thrombin cleavable linker sequence and the N-terminalportion of a core streptokinase molecule, was gel purified and ligatedto the vector DNA of pGC606 of Example 5 after treatment with HindIIIand phosphatase. The recombinant ligation products were transformed intocompetent cells of E. coli strain HW1110 (lacIq). Ampicillin (100 μg/ml)resistant transformants were analysed by zymography as described inExample 11 below. A correct clone pLGC4, was identified.

EXAMPLE 8 Construction of a Factor Xa-CleavableHirudin-IEGR-Streptokinase Fusion Gene

A hirudin-streptokinase fusion has been designed in which a full lengthhirudin molecule is joined to full length streptokinase via an IEGRlinker sequence cleavable by factor Xa; see SEQ ID: NOS:42 and 43 Thegene encoding the hirudin-streptokinase protein was constructed by sitedirected mutagenesis and molecular cloning. In order to juxtapose thehirudin and streptokinase genes, the DNA fragments encoding these geneswere ligated together. The streptokinase gene from plasmid pKJ2 ofExample 4 was isolated by gel purification of a ca. 1.4 kbp DNA fragmentafter digestion of pKJ2 vector DNA with BqlII and BamHI. This DNAfragment contains all of the streptokinase gene together with the DNAencoding the streptokinase signal peptide sequence. This DNA fragmentwas then ligated to BamHI treated pJK1 DNA of Preparation 2 whichcontains the hirudin encoding DNA sequence. The recombinant ligationproducts were transformed into competent cells of E. coli strain HW1110(lacIq). Ampicillin (100 μg/ml) resistant transformants were screened bypreparation of plasmid DNA, restriction endonuclease digestion withHindIII and agarose gel electrophoresis. There are two possibleorientations for the insert in this cloning event and correct cloneswere identified as those which released a ca. 1080 bp DNA fragment afterHindIII digestion as analysed on agarose gels. One such clone pJK3,which contains the hirudin gene separated from the streptokinase gene bythe streptokinase signal peptide sequence, was used in subsequentmanipulations. To create a template for mutagenesis to delete theintervening sequences and to insert the DNA encoding the factor Xacleavable linker sequence, the hirudin-streptokinase portion of pJK3 wastransferred to a mutagenesis vector M13mp18. Plasmid DNA of pJK3 wasdigested with KpnI and BamHI and the ca. 1490 bp DNA fragment gelpurified and ligated to KpnI and BamHI treated M13mp18 replicative formDNA. The recombinant ligation products were transfected into competentcells of E. coli JM103 (Example 1). Single stranded DNA was preparedfrom putative recombinant plaques and a correct clone pSMD1/100 (1.1 )was identified. To delete the streptokinase signal peptide sequence andto insert the DNA encoding the factor Xa linker sequence single strandedDNA of pSMD1/100 (1.1) was used as a template for mutagenesis with a46-mer oligonucleotide BB3317:(5'-CACTCAGGTCCAGCAATTCTACCTTCGATCTGCAGATATTCTTCTG-3') SEQ ID: NO:44.Single stranded DNA from putative mutant plaques were prepared and amutant pGC615 was identified by DNA sequence analysis using thesequencing primer BB3510 (5'-CACTATCAGTAGCAT-3') SEQ ID: NO:45. pGC615contains the C-terminal portion of the hirudin gene linked to the maturestreptokinase protein coding sequence. In order to reconstruct thehirudin gene, replicative form DNA of pGC615 was treated with KpnI andBamHI, the ca. 1320 bp DNA fragment gel purified and ligated to KpnI andBamHI treated pJC80 of Preparation 2. The recombinant ligation productswere transformed into competent cells of E. coli strain DH5 (Example 4).Ampicillin (100 μg/ml) resistant transformants were screened bypreparation of plasmid DNA, restriction endonuclease analysis with KpnI,BamHI and HindIII and agarose gel electrophoresis. A clone with thecorrect electrophoretic pattern pSMD1/139 was identified. This plasmidcontains DNA encoding the complete factor Xa cleavablehirudin-streptokinase fusion molecule.

EXAMPLE 9 Construction of a Vector for the Expression of a Factor XaCleavable Hirudin-IEGR-Streptokinase Fusion Molecule

To construct a vector for the expression of thehirudin-IEGR-streptokinase gene, DNA of pSMD1/139 of Example 8 wastreated with HindIII and a ca. 963 bp DNA fragment encoding part of theyeast alpha factor secretion signal, all of hirudin, the factor Xalinker and the 5' part of streptokinase as far as the internal HindIIIsite in the streptokinase sequence was gel purified. This fragment wasthen ligated to HindIII treated and phosphatased DNA of pSMD1/119 ofExample 4. The recombinant ligation products were transformed intocompetent cells of E. coli strain DH5 (Example 4). Ampicillin resistanttransformants were screened by preparation of plasmid DNA, restrictionendonuclease digestion with KpnI and BamHI and agarose gelelectrophoresis. It is possible to obtain two orientations of theHindIII insert and one clone in the correct orientation pSMD1/146 wasidentified as releasing a ca. 1311 bp fragment after KpnI and BamHItreatment. pSMD1/146 contains the full length fusion gene under thecontrol of the regulatable PAL promoter described in Preparation 2, andhas been designed for the regulated expression and secretion of thefactor Xa-cleavable hirudin-streptokinase fusion protein. pSMD1/146plasmid DNA was prepared and used to transform yeast strain BJ2168(Preparation 3) according to the method of Sherman, F. et al., (Methodsin Yeast Genetics, Cold Spring Harbor Laboratory (1986)).

EXAMPLE 10 Construction of a Factor Xa CleavableStreptokinase-IEGR-Hirudin Fusion Gene and its Expression Vector

A gene encoding a streptokinase-hirudin fusion protein linked via aFactor Xa cleavage site (IEGR) was constructed by site-directedmutagenesis and molecular cloning SEQ ID: NOS:46 and 47 In order tojuxtapose the streptokinase and hirudin genes, DNA fragments encodingthese two gene were ligated together. The pUC19SK vector DNA ofPreparation 4 was prepared and treated with HindIII and BamHI and theca. 500 bp DNA fragment containing the 3' end of the streptokinase genewas gel purified. This fragment was ligated to M13mp19 replicative formDNA treated with HindIII and BamHI. The recombinant ligation mixture wastransfected into competent cells of E. coli strain JM103 (Example 1).Single stranded DNA was prepared from putative recombinant plaques andthe required clone M13JK1 identifed by dideoxy sequence analysis usingthe M13 universal sequencing primer (SEQ ID: NO:11, Example 1). M13JK1contains the C-terminal portion of the streptokinase gene. The α-factorhirudin gene was then cloned into M13JK1 to juxtapose both sequences.Plasmid DNA of pJK1 of Preparation 2 was digested with BqlII and BamHIand a ca. 465bp DNA fragment encoding the α-factor hirudin fusion wasgel purified. This DNA fragment was then ligated to BamHI treatedreplicative form DNA of M13JK1. The recombinant ligation products weretransfected into competent cells of E. coli strain JM103. Singlestranded DNA from putative recombinant plaques were prepared and acorrect clone SMD1/100.3 identified by dideoxy sequence analysis usingM13 universal sequencing primer (SEQ ID: NO:11, Example 1. SMD1/100.3contains the C-terminal portion of the streptokinase gene and thecomplete hirudin gene separated by the α-factor encoding sequencedescribed in Preparation 2. In order to delete this sequence and replaceit with a factor Xa-cleavable linker sequence, SMD1/100.3 was used as atemplate for site-directed mutagenesis. Single stranded DNA ofSMD1/100.3 was prepared and used for mutagenesis using a 47-mermutagenesis primer BB3318:(5'-TCGGTGTAAACAACTCTTCTACCTTCGATTTTGTCGTTAGGGTTATC-3") (SEQ ID: NO:49).Single stranded DNA from putative mutant plaques were prepared and therequired mutation pGC616 identified by dideoxy sequence analysis usingthe sequencing primer BB2018:(5'-GCGGCTTTGGGGTACCTTCACCAGTGACACATTGG-3') (SEQ ID: NO:57). pGC616contains an additional mutation inadvertently introduced by themutagenesis procedure. This was corrected by a further mutagenic step.Single stranded DNA of pGC616 was prepared and used as a template formutagenesis with a 21-mer oligonucleotide BB3623(5'-GTGTAAACAACTCTACCTTCG-3') (SEQ ID: NO:49). Single stranded DNA fromputative mutant plaques was prepared and a correct clone pGC620identified by dideoxy sequence analysis with the sequencing primerBB2018 (SEQ ID: NO:57). pGC620 contains the C-terminal portion of thestreptokinase gene and the complete hirudin gene fused via DNA encodinga factor Xa-cleavable linker. The intact factor Xa-cleavablestreptokinase-hirudin fusion gene was reconstructed in two steps. TheC-terminal streptokinase-hirudin sequence from pGC620 was cloned intothe yeast expression vector pSW6 of Preparation 2 and then theN-terminal portion of streptokinase was cloned into the new vector tocreate the full length streptokinase-hirudin fusion gene.

Replicative form DNA of pGC620 was treated with HindIII and BamHI and aca. 710 bp HindIII-BamHI DNA fragment encoding the 3' end ofstreptokinase, the intervening factor Xa-cleavable linker DNA sequenceand all of the hirudin gene was gel purified. This ca. 710 bp DNAfragment was ligated to pSW6 of Preparation 2 digested with HindIII andBamHI. The recombinant ligation products were transformed into competentcells of E. coli strain DH5 (Example 4). Ampicillin (100 μg/ml)resistant transformants were screened by preparation of plasmid DNA,restriction endonuclease analysis using HindIII and BamHI and agarosegel electrophoresis. A clone with the correct electrophoretic patternpSMD1/143 was identified. The intact fusion gene was then constructed bycloning the N-terminal portion of α-factor-streptokinase into pSMD1/143.Replicative form DNA of pGC614 of Example 4 was treated with HindIII andthe ca. 750 bp DNA fragment containing the N-terminal portion ofα-factor-streptokinase gel purified and ligated to HindIII treated andphosphatased pSMD1/143 vector DNA. The recombinant ligation productswere transformed into competent cells of E. coli strain DH5. Ampicillin(100 μg/ml) resistant transformants were screened by preparation ofplasmid DNA, restriction endonuclease digestion with DraI and agarosegel electrophoresis. A clone in the correct orientation pSMD1/159 wasidentified as giving rise to 4 fragments of sizes of about 4750 bp, 2140bp, 1526 bp, and 692 bp after DraI digestion. pSMD1/159 was used for theexpression of the factor Xa-cleavable streptokinase-hirudin fusionprotein. pSMD1/159 plasmid DNA was prepared and used to transform yeaststrain BJ2168 (Preparation 5) according to the method of Sherman, F. etal., (Methods in Yeast Genetics, Cold Spring Harbour Laboratory (1986)).

EXAMPLE 11 Expression of Monomer Streptokinase Constructs Expression

Competent cells of E. coli strain JM103 (Example 1) were transformedwith DNA of the streptokinase expression vectors of Examples 4, 5, 6 and7. The lacIq gene in the expression host is desirable to represstranscription from the tac promoter used in all of the E. coliexpression constructs. All media for the growth of recombinant E. colistrains were as described in Maniatis et al. Using 1 liter shake flasks,cultures of recombinant E. coli containing streptokinase expressionvectors were grown in 250 ml batches of 2TY medium containing 100 μg/mlof carbenicillin at 37° C. in an orbital shaker. The optical density ofthe cultures were monitored at 600 nm. When the culture reached an OD600 nm of 0.5, expression from the tac promoter was induced by theaddition of isopropyl-β-D-thiogalactoside (IPTG) to a finalconcentration of 1 mM. After growth for 30 to 240 min the cells wereharvested by centrifugation. SDS-PAGE Separation

The ability of the recombinant E. coli cells to express streptokinasewas assayed using zymography. The quantity and molecular weight ofstreptokinase activity of an E. coli culture was estimated by thefollowing protocol. A 1 ml aliquot of the culture was removed, the cellswere harvested by centrifugation (14 000×g) for 5 mins and resuspendedin 200 μl of loading buffer (125 mM Tris.HCl pH 6.8, 10% glycerol (w/v),0.01% (w/v) bromophenol blue, 1% (v/v) 2-mercaptoethanol, 6M urea). Analiquot of this mixture was applied to an SDS-PAGE gel and the proteinseparated by electrophoresis. The quantity of protein loaded onto thegel was varied by altering the size of the aliquot according to theoptical density of the culture upon harvesting. Generally, 10 μl of themixture from a culture of OD 600 nm of 1.0 was used for each lane.

Zymography

After electrophoresis the polyacrylamide gel was washed in 2% (w/v)Triton X-100 (3×20 mins) followed by water washes (3×20 mins) to removethe SDS and allow renaturation of the streptokinase molecule.

The washed SDS-PAGE gel was then overlayed with an agarose mixtureprepared as follows. 200 mg of agarose was dissolved in 18 ml distilledand deionised water (dH₂ O ) and allowed to cool to 55°-60° C. To this200 mg of MARVEL (trade mark of Premier Brands, U.K. Ltd. P.O. Box 171,Birmingham, B30 2NA, U.K.) (casein) dissolved in 2 ml of dH₂ O, ml of 1MTris.HCl pH 8.0 and 600 μl of 5M NaCl were added. Just before pouringover the washed SDS-PAGE gel, 700 μl of plasminogen at 300 μg/ml (SigmaP-7397 10 mg/ml in 50 mM Tris. HCl pH 7.5) was added and mixedthoroughly. The mixture was poured over the gel and once set wasincubated at 37° C. for 2 hours when it could be inspected. Plasminogenactivator activity (streptokinase activity) was detected by plasmindigestion of the opaque casein containing overlay which produced clearzones. The position of the zones on the gel was directly related to thesize of the active molecules.

The recombinant E. coli JM103 strains containing monomer streptokinaseexpression vectors pKJ2 of Example 4 and pLGC1 of Example 4 bothexpressed streptokinase activity with a molecular weight ofapproximately 47 kDa (FIG. 5).

EXAMPLE 12 Expression of a Thrombin CleavableStreptokinase-Streptokinase Fusion Protein.

A recombinant E. coli HW1110 (lacIq) strain (Example 1) containing pLGC2of Example 6, the thrombin cleavable streptokinase- streptokinase fusiongene, was expressed and analysed according to the expression andzymography protocols of Example 11. The E. coli JM103/pLGC2 strainexpressed streptokinase activities of several molecular weights,predominantly of 110 kDa and 47 kDa (FIG. 5). Cleavage analysis isdescribed in Example 13 below.

EXAMPLE 13 Cleavage of the Thrombin CleavableStreptokinase-streptokinase Fusion Protein by Thrombin

Using 1 liter shake flasks, a 3 liter culture of E. coli JM103(Example 1) containing pLGC2 of Example 6 was grown in 500 ml batches in2TY medium containing 100 mcg/ml carbenicillin at 37° C. with vigorousshaking in an orbital shaker. When the optical density of the culturesreached an O.D. 600 nm of 0.5 the expression of thestreptokinase-streptokinase fusion protein was induced by the additionof IPTG to a final concentration of 1 mM. The cultures were incubated at37° C. with vigorous shaking for a further 4 hours when they wereharvested by centrifugation at 8,000 r.p.m. for 10 mins. The cells wereresuspended in 10 ml of ice cold TS buffer (10 mM Tris.HCl pH 7.5, 20%(w/v) sucrose). 348 μl of 0.5M EDTA was added and the mixture incubatedon ice for 10 mins. The cells were harvested by centrifugation at 8,000r.p.m. for 5 min at 4° C. and the supernatant discarded. The cells wereresuspended in 6.25 ml of ice cold sterile H₂ O and incubated on ice for10 min. The cells were harvested by centrifugation at 8,000 rpm. for 5min at 15,000 g for 30 min at 4° C. and the supernatant discarded. Thecells were resuspended in 48 ml of ARG buffer (20 mM Tris.HCl pH 7.5, 10mM MgCl₂, 10 mM 2-b-mercaptoethanol, 0.5 mM phenylmethyl sulphonylfluoride, 12 mcM N-tosyl-L-phenylalanine chloromethyl ketone) andsonicated on ice (6×30 sec. bursts on maximum power, MSE SONIPREP 150(trade mark)). The cell sonicate was centrifuged at 15,000 g for 30 minat 4° C. The supernatant was decanted and assayed for streptokinaseactivity using the S2251 (KabiVitrum Ltd, KabiVitrum House, RiversideWay, Uxbridge, Middlesex, UB8 2YF, UK) chromogenic assay for thestreptokinase activation of plasminogen. S2251 is a specific tripeptidechromogenic substrate for plasmin. 25 μl of 0.1M Tris. HCl pH 8.0 wasplaced in wells 2 to 12 of 96 well plates. Aliquots of the sample (25 μl) were placed in wells 1 and 2, and two-fold dilutions made by mixingand pipetting from wells 2 to 3, 3 to 4 and so on to well 11. A 100 μlaliquot of a plasminogen/S2251 mixture (40 μl plasminogen 300 μg/ml, 220μl S2251 1 mg/ml, 1.04 ml 0.1M Tris. HCl pH 7.5) was added to each welland the plate incubated at 37° C. for 30 min. The reaction wasterminated by the addition of 50 mcl of 0.5M acetic acid. The absorbancewas read at 405 nM using an automatic plate reader. Quantification wasperformed by comparison with a standard streptokinase preparation. Theanalysis showed that the supernatant contained approximately 2560 u/mlof streptokinase activity.

Solid ammonium sulphate was slowly added to the supernatant to 15%saturation (4.03 g) and allowed to dissolve for 15 min at roomtemperature. The mixture was then centrifuged for 30 min at 15,000 g atroom temperature. The supernatant was decanted and additional solidammonium sulphate was added to 40% saturation (7.27 g), and allowed todissolve. The mixture was centrifuged for 30 min at 15,000 g at roomtemperature and the supernatant discarded. The pelleted protein (the15-40% cut), was resuspended in 10 ml of ARG buffer. A portion of the15-40% cut was assayed using the S2251 chromgenic assay and was found tocontain 18,432 u/ml of streptokinase activity.

The ability of thrombin to cleave the streptokinase-streptokinase fusionprotein at the thrombin cleavable linker was assessed by an in vitrocleavage assay and zymography. A 5 μl aliquot of the 15-40% cut wasmixed with 45 μl of ARG buffer to dilute the protein ten-fold. 10 μl ofthis mixture was incubated with 5 u/ml of thrombin in a final volume of50 μl at 37° C. for 14 hours. Aliquots (10 μl ) of the thrombin cleavagereactions were analysed by zymography according to the method of Example11. The results are shown in FIG. 6. The streptokinase-streptokinasefusion protein (Mr 110 kDa), was quantitatively cleaved whilst the lowermolecular weight streptokinase activity (Mr 47 kDa) was not cleaved bythrombin. Thus the streptokinase-streptokinase fusion protein iscleavable by thrombin.

EXAMPLE 14 Expression of a Factor Xa CleavableStreptokinase-IEGR-hirudin Fusion Gene

Plasmid expression vector pSMD1/159 of Example 10 was transferred intoyeast (S. cerevisiae) strain BJ2168 according to the method ofPreparation 3. Using 500 ml shake flasks, cultures of yeast containingpSMD1/159 were grown in 100 ml batches of 0.67% synthetic completemedium yeast nitrogen base, with amino acids minus leucine and 1%glucose as a carbon source. After overnight growth at 30° C., the cellswere harvested by centrifugation at 3,000 rpm for 10 min and resuspendedin the same synthetic complete medium except having 1% galactose and0.2% glucose as the carbon source and the addition of sodium phosphate(to 50 mM) pH 7.2. This induces the expression of thestreptokinase-hirudin fusion gene from the hybrid PGK promoter. Cellswere grown in the induction medium for 3 days. After this period, thesupernatant was harvested by centrifugation. The broth was assayed forboth streptokinase activity according to the S2251 assay procedure ofExample 13 and hirudin activity according to the thrombin inhibitionassay of Example 2. Both activities were detected and secreted to themedium.

EXAMPLE 15 Expression of a Factor Xa CleavableHirudin-IEGR-Streptokinase Fusion Gene

Plasmid expression vector pSMD1/146 of Example 9 was transferred intoyeast (S. cerevisiae) strain BJ2168 according to the method ofPreparation 3. The culture was incubated, expressed, harvested and thehirudin and streptokinase activities assayed according to the methods ofExamples 2 and 13. Both streptokinase and hirudin activities weredetected and secreted to the medium.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 73                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 201 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..201                                                          (D) OTHER INFORMATION: /note="hirudin type HV-1 gene"                         (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..195                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..195                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTTGTTTACACCGACTGTACTGAATCCGGA CAAAACCTGTGTTTGTGT48                           ValValTyrThrAspCysThrGluSerGlyGlnAsnLeuCysLeuCys                              151015                                                                        GAGGGTTCTAACGTCTGTGGTCAGGGTAAC AAATGCATCCTGGGTTCC96                           GluGlySerAsnValCysGlyGlnGlyAsnLysCysIleLeuGlySer                              202530                                                                        GACGGTGAAAAGAACCAATGTGTCACTGGTGA AGGTACCCCAAAGCCG144                          AspGlyGluLysAsnGlnCysValThrGlyGluGlyThrProLysPro                              354045                                                                        CAGTCCCACAACGATGGAGATTTCGAAGAAATCCCAG AAGAATATCTG192                          GlnSerHisAsnAspGlyAspPheGluGluIleProGluGluTyrLeu                              505560                                                                        CAGTAATAG 201                                                                 Gln                                                                           65                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ValValTyrThrAspCysThrGluSerGlyGlnAsnLeuCysLeuCys                               151015                                                                       GluGlySerAsnValCysGlyGlnGlyAsnLysCysIleLeuGlySer                              202530                                                                        AspGlyGluLys AsnGlnCysValThrGlyGluGlyThrProLysPro                             354045                                                                        GlnSerHisAsnAspGlyAspPheGluGluIleProGluGluTyrLeu                              5055 60                                                                       Gln                                                                           65                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: /note="oligonucleotide universal                       primer for pUC19"                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CAGGGTTTTCCCAGTCACG19                                                         (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7859 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: circular                                                       (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..7859                                                         (D) OTHER INFORMATION: /note="sequence of plasmid pSW6"                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TTCCCATGTCTCTACTGGTGGTGGTGCTTCTTTGGAATTATTGGAAGGTAAGGAATTGCC60                AGGTG TTGCTTTCTTATCCGAAAAGAAATAAATTGAATTGAATTGAAATCGATAGATCAA120              TTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTG180               AATATTTTTTATTTATATACGTATATATAGACTATTATTTACTTTTAATA GATTATTAAG240              ATTTTTATTAAAAAAAAATTCGTCCCTCTTTTTAATGCCTTTTATGCAGTTTTTTTTTCC300               CATTCGATATTTCTATGTTCGGGTTTCAGCGTATTTTAAGTTTAATAACTCGAAAATTCT360               GCGTTTCGAAAAAGCTCTGCATTAATGA ATCGGCCAACGCGCGGGGAGAGGCGGTTTGCG420              TATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCG480               GCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAA540               CGCAG GAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC600              GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTC660               AAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTC CCCCTGGAAG720              CTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCT780               CCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTA840               GGTCGTTCGCTCCAAGCTGGGCTGTGTG CACGAACCCCCCGTTCAGCCCGACCGCTGCGC900              CTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGC960               AGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTT1020              GAAGT GGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT1080             GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGC1140              TGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAA AAGGATCTCA1200             AGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTA1260              AGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAA1320              ATGAAGTTTTAAATCAATCTAAAGTATA TATGAGTAAACTTGGTCTGACAGTTACCAATG1380             CTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTG1440              ACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGC1500              AATGA TACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGC1560             CGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAA1620              TTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCA ACGTTGTTGC1680             CATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGG1740              TTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTC1800              CTTCGGTCCTCCGATCGTTGTCAGAAGT AAGTTGGCCGCAGTGTTATCACTCATGGTTAT1860             GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGG1920              TGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC1980              GGCGT CAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGG2040             AAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGAT2100              GTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCA GCGTTTCTGG2160             GTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATG2220              TTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCT2280              CATGAGCGGATACATATTTGAATGTATT TAGAAAAATAAACAAATAGGGGTTCCGCGCAC2340             ATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTA2400              TAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTGAACCAGTCCTAAAAC2460              GAGTA AATAGGACCGGCAATTCTTCAAGCAATAAACAGGAATACCAATTATTAAAAGATA2520             ACTTAGTCAGATCGTACAATAAAGCTAGCTTTGAAGAAAAATGCGCCTTATTCAATCTTT2580              GCTATAAAAAATGGCCCAAAATCTCACATTGGAAGACATTTGATGACCTC ATTTCTTTCA2640             ATGAAGGGCCTAACGGAGTTGACTAATGTTGTGGGAAATTGGAGCGATAAGCGTGCTTCT2700              GCCGTGGCCAGGACAACGTATACTCATCAGATAACAGCAATACCTGATCACTACTTCGCA2760              CTAGTTTCTCGGTACTATGCATATGATC CAATATCAAAGGAAATGATAGCATTGAAGGAT2820             GAGACTAATCCAATTGAGGAGTGGCAGCATATAGAACAGCTAAAGGGTAGTGCTGAAGGA2880              AGCATACGATACCCCGCATGGAATGGGATAATATCACAGGAGGTACTAGACTACCTTTCA2940              TCCTA CATAAATAGACGCATATAAGTACGCATTTAAGCATAAACACGCACTATGCCGTTC3000             TTCTCATGTATATATATATACAGGCAACACGCAGATATAGGTGCGACGTGAACAGTGAGC3060              TGTATGTGCGCAGCTCGCGTTGCATTTTCGGAAGCGCTCGTTTTCGGAAA CGCTTTGAAG3120             TTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAGCGCTTTTGAAAA3180              CCAAAAGCGCTCTGAAGACGCACTTTCAAAAAACCAAAAACGCACCGGACTGTAACGAGC3240              TACTAAAATATTGCGAATACCGCTTCCA CAAACATTGCTCAAAAGTATCTCTTTGCTATA3300             TATCTCTGTGCTATATCCCTATATAACCTACCCATCCACCTTTCGCTCCTTGAACTTGCA3360              TCTAAACTCGACCTCTACATTTTTTATGTTTATCTCTAGTATTACTCTTTAGACAAAAAA3420              ATTGT AGTAAGAACTATTCATAGAGTGAATCGAAAACAATACGAAAATGTAAACATTTCC3480             TATACGTAGTATATAGAGACAAAATAGAAGAAACCGTTCATAATTTTCTGACCAATGAAG3540              AATCATCAACGCTATCACTTTCTGTTCACAAAGTATGCGCAATCCACATC GGTATAGAAT3600             ATAATCGGGGATGCCTTTATCTTGAAAAAATGCACCCGCAGCTTCGCTAGTAATCAGTAA3660              ACGCGGGAAGTGGAGTCAGGCTTTTTTTATGGAAGAGAAAATAGACACCAAAGTAGCCTT3720              CTTCTAACCTTAACGGACCTACAGTGCA AAAAGTTATCAAGAGACTGCATTATAGAGCGC3780             ACAAAGGAGAAAAAAAGTAATCTAAGATGCTTTGTTAGAAAAATAGCGCTCTCGGGATGC3840              ATTTTTGTAGAACAAAAAAGAAGTATAGATTCTTTGTTGGTAAAATAGCGCTCTCGCGTT3900              GCATT TCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGC3960             GTTGCATTTTTGTTTTACAAAAATGAAGCACAGATTCTTCGTTGGTAAAATAGCGCTTTC4020              GCGTTGCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAA AATTAGCGCT4080             CTCGCGTTGCATTTTTGTTCTACAAAATGAAGCACAGATGCTTCGTTAACAAAGATATGC4140              TATTGAAGTGCAAGATGGAAACGCAGAAAATGAACCGGGGATGCGACGTGCAAGATTACC4200              TATGCAATAGATGCAATAGTTTCTCCAG GAACCGAAATACATACATTGTCTTCCGTAAAG4260             CGCTAGACTATATATTATTATACAGGTTCAAATATACTATCTGTTTCAGGGAAAACTCCC4320              AGGTTCGGATGTTCAAAATTCAATGATGGGTAACAAGTACGATCGTAAATCTGTAAAACA4380              GTTTG TCGGATATTAGGCTGTATCTCCTCAAAGCGTATTCGAATATCATTGAGAAGCTGC4440             ATTTTTTTTTTTTTTTATATATATTTCAAGGATATACCATTGTAATGCCTGCCCCTAAGA4500              AGATCGTCGTTTTGCCAGGTGACCACGTTGGTCAAGAAATCACAGCCGAA GCCATTAAGG4560             TTCTTAAAGCTATTTCTGATGTTCGTTCCAATGTCAAGTTCGATTTCGAAAATCATTTAA4620              TTGGTGGTGCTGCTATCGATGCTACAGGTGTTCCACTTCCAGATGAGGCGCTGGAAGCCT4680              CCAAGAAGGCTGATGCCGTTTTGTTAGG TGCTGTGGGTGGTCCTAAATGGGGTACCGGTA4740             GTGTTAGACCTGAACAAGGTTTACTAAAAATCCGTAAAGAACTTCAATTGTACGCCAACT4800              TAAGACCATGTAACTTTGCATCCGACTCTCTTTTAGACTTATCTCCAATCAAGCCACAAT4860              TTGCT AAAGGTACTGACTTCGTTGTTGTTAGAGAATTAGTGGGAGGTATTTACTTTGGTA4920             AGAGAAAGGAAGACGATGGTGATGGTGTCGCTTGGGATAGTGAACAATACACCGTTCCAG4980              AAGTGCAAAGAATCACAAGAATGGCCGCTTTCATGGCCCTACAACATGAG CCACCATTGC5040             CTATTTGGTCCTTGGATAAAGCTAATGTTTTGGCCTCTTCAAGATTATGGAGAAAAACTG5100              TGGAGGAAACCATCAAGAACGAATTCCCTACATTGAAAGTTCAACATCAATTGATTGATT5160              CTGCCGCCATGATCCTAGTTAAGAACCC AACCCACCTAAATGGTATTATAATCACCAGCA5220             ACATGTTTGGTGATATCATCTCCGATGAAGCCTCCGTTATCCCAGGCTCCTTGGGTTTGT5280              TGCCATCTGCGTCCTTGGCCTCTTTGCCAGACAAGAACACCGCATTTGGTTTGTACGAAC5340              CATGC CATGGTTCCGCTCCAGATTTGCCAAAGAATAAGGTCAACCCTATCGCCACTATCT5400             TGTCTGCTGCAATGATGTTGAAATTGTCATTGAACTTGCCTGAAGAAGGTAAAGCCATTG5460              AAGATGCAGTTAAAAAGGTTTTGGATGCAGGTATCAGAACTGGTGATTTA GGTGGTTCCA5520             ACAGTACCACCGAAGTCGGTGATGCTGTCGCCGAAGAAGTTAAGAAAATCCTTGCTTAAA5580              AAGATTCTCTTTTTTTATGATATTTGTACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA5640              AAAAAAAAAAAAAAAAAAAAAAAATGCA GCGTCACATCGGATAATAATGATGGCAGCCAT5700             TGTAGAAGTGCCTTTTGCATTTCTAGTCTCTTTCTCGGTCTAGCTAGTTTTACTACATCG5760              CGAAGATAGAATCTTAGATCACACTGCCTTTGCTGAGCTGGATCAATAGAGTAACAAAAG5820              AGTGG TAAGGCCTCGTTAAAGGACAAGGACCTGAGCGGAAGTGTATCGTACAGTAGACGG5880             AGTATACTAGTATAGTCTATAGTCCGTGGAATTCTCATGTTTGACAGCTTATCATCGATA5940              AGCTAGCTTTCTAACTGATCTATCCAAAACTGAAAATTACATTCTTGATT AGGTTTATCA6000             CAGGCAAATGTAATTTGTGGTATTTTGCCGTTCAAAATCTGTAGAATTTTCTCATTGGTC6060              ACATTACAACCTGAAAATACTTTATCTACAATCATACCATTCTTAATAACATGTCCCCTT6120              AATACTAGGATCAGGCATGAACGCATCA CAGACAAAATCTTCTTGACAAACGTCACAATT6180             GATCCCTCCCCATCCGTTATCACAATGACAGGTGTCATTTTGTGCTCTTATGGGACGATC6240              CTTATTACCGCTTTCATCCGGTGATTGACCGCCACAGAGGGGCAGAGAGCAATCATCACC6300              TGCAA ACCCTTCTATACACTCACATCTACCAGTGATCGAATTGCATTCAGAAAACTGTTT6360             GCATTCAAAAATAGGTAGCATACAATTAAAACATGGCGGGCATGTATCATTGCCCTTATC6420              TTGTGCAGTTAGACGCGAATTTTTCGAAGAAGTACCTTCAAAGAATGGGG TCTTATCTTG6480             TTTTGCAAGTACCACTGAGCAGGATAATAATAGAAATGATAATATACTATAGTAGAGATA6540              ACGTCGATGACTTCCCATACTGTAATTGCTTTTAGTTGTGTATTTTTAGTGTGCAAGTTT6600              CTGTAAATCGATTAATTTTTTTTTCTTT CCTCTTTTTATTAACCTTAATTTTTATTTTAG6660             ATTCCTGACTTCAACTCAAGACGCACAGATATTATAACATCTGCATAATAGGCATTTGCA6720              AGAATTACTCGTGAGTAAGGAAAGAGTGAGGAACTATCGCATACCTGCATTTAAAGATGC6780              CGATT TGGGCGCGAATCCTTTATTTTGGCTTCACCCTCATACTATTATCAGGGCCAGAAA6840             AAGGAAGTGTTTCCCTCCTTCTTGAATTGATGTTACCCTCATAAAGCACGTGGCCTCTTA6900              TCGAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAA CTGAAAAAAC6960             CCAGACACGCTCGACTTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAAC7020              AAGGTCCTAGCGACGGCTCACAGGTTTTGTAACAAGCAATCGAAGGTTCTGGAATGGCGG7080              GGAAAGGGTTTAGTACCACATGCTATGA TGCCCACTGTGATCTCCAGAGCAAAGTTCGTT7140             CGATCGTACTGTACTCTCTCTCTTTCAAACAGAATTGTCCGAATCGTGTGACAACAACAG7200              CCTGTTCTCACACACTCTTTTCTTCTAACCAAGGGGGTGGTTTAGTTTAGTAGAACCTCG7260              TGAAA CTTACATTTACATATATATAAACTTGCATAAATTGGTCAATGGAAGAAATACATA7320             TTTGGTCTTTTCTAATTCGTAGTTTTTCAAGTTCTTAGATGCTTTCTTTTTCTCTTTTTT7380              ACAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAATACAAAAGA TCTATGAGAT7440             TTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCA7500              ACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTTAG7560              ATTTAGAAGGGGATTTCGATGTTGCTGT TTTGCCATTTTCCAACAGCACAAATAACGGGT7620             TATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTAAGCTTGG7680              ATAAAAGAAACAGCGACTCTGAATGCCCGCTGAGCCATGATGGCTACTGCCTGCACGACG7740              GTGTA TGCATGTATATCGAAGCTCTGGACAAATACGCATGCAACTGCGTAGTTGGTTACA7800             TCGGCGAACGTTGCCAGTACCGCGACCTGAAATGGTGGGAGCTCCGTTAATAAGGATCC7859               (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B ) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note="oligonucleotide top strand                      adapter to fuse C-terminal end of the a-factor                                pro-peptide to synthetic hirudin gene"                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AGCTTGGATA AAAGA15                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: /note="bottom strand of adapter to                     fuse c- terminal end of the a-factor pro-peptide to                           synthetic hirudin gene"                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TCTTTTATCCA11                                                                 (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 223 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..223                                                          (D) OTHER INFORMATION: /note="hirudin type HV-1 gene with                     5 amino acid adapter (corresponding to C-terminus                              of alpha factor) at amino terminus"                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note="HindIII site"                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 218..223                                                        (D) OTHER INFORMATION: /note="BamHI site"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AAGCTTGGATAAAAGAGTTGTTT ACACCGACTGTACTGAATCCGGACAAAACCTGTGTTT60               GTGTGAGGGTTCTAACGTCTGTGGTCAGGGTAACAAATGCATCCTGGGTTCCGACGGTGA120               AAAGAACCAATGTGTCACTGGTGAAGGTACCCCAAAGCCGCAGTCCCACAACGATGGAGA180                TTTCGAAGAAATCCCAGAAGAATATCTGCAGTAATAGGGATCC223                               (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 420 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                            (B) LOCATION: 1..420                                                          (D) OTHER INFORMATION: /note="Factor Xa-cleavable                             Hirudin-IEGR- Hirudin"                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..402                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..402                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GTTGTTTACAC CGACTGTACTGAATCCGGACAAAACCTGTGTTTGTGT48                           ValValTyrThrAspCysThrGluSerGlyGlnAsnLeuCysLeuCys                              151015                                                                        GAGGGTTCTA ACGTCTGTGGTCAGGGTAACAAATGCATCCTGGGTTCC96                           GluGlySerAsnValCysGlyGlnGlyAsnLysCysIleLeuGlySer                              202530                                                                        GACGGTGAAAAG AACCAATGTGTCACTGGTGAAGGTACCCCAAAGCCG144                          AspGlyGluLysAsnGlnCysValThrGlyGluGlyThrProLysPro                              354045                                                                        CAGTCCCACAACGATGGA GATTTCGAAGAAATCCCAGAAGAATATCTG192                          GlnSerHisAsnAspGlyAspPheGluGluIleProGluGluTyrLeu                              505560                                                                        CAGATCGAAGGAAGAGTTGTTTACAC CGACTGTACTGAATCCGGACAA240                          GlnIleGluGlyArgValValTyrThrAspCysThrGluSerGlyGln                              65707580                                                                      AACCTGTGTTTGTGTGAGGGTT CTAACGTCTGTGGTCAGGGTAACAAA288                          AsnLeuCysLeuCysGluGlySerAsnValCysGlyGlnGlyAsnLys                              859095                                                                        TGCATCCTGGGTTCCGACGGT GAAAAGAACCAATGTGTCACTGGTGAA336                          CysIleLeuGlySerAspGlyGluLysAsnGlnCysValThrGlyGlu                              100105110                                                                     GGTACCCCAAAGCCGCAGTCCCAC AACGATGGAGATTTCGAAGAAATC384                          GlyThrProLysProGlnSerHisAsnAspGlyAspPheGluGluIle                              115120125                                                                     CCAGAAGAATATCTGCAGTAATAGGGATCCG AATTC420                                      ProGluGluTyrLeuGln                                                            130                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 134 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ValValTyrThrAsp CysThrGluSerGlyGlnAsnLeuCysLeuCys                             151015                                                                        GluGlySerAsnValCysGlyGlnGlyAsnLysCysIleLeuGlySer                              20 2530                                                                       AspGlyGluLysAsnGlnCysValThrGlyGluGlyThrProLysPro                              354045                                                                        GlnSerHisAsnAspGlyAspPheGluGluIleProGlu GluTyrLeu                             505560                                                                        GlnIleGluGlyArgValValTyrThrAspCysThrGluSerGlyGln                              65707580                                                                      AsnL euCysLeuCysGluGlySerAsnValCysGlyGlnGlyAsnLys                             859095                                                                        CysIleLeuGlySerAspGlyGluLysAsnGlnCysValThrGlyGlu                              1 00105110                                                                    GlyThrProLysProGlnSerHisAsnAspGlyAspPheGluGluIle                              115120125                                                                     ProGluGluTyrLeuGln                                                            130                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..46                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2988"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CAGT CGGTGTAAACAACTCTTCCTTCGATCTGCAGATATTCTTCTG46                             (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                            (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: /note="M13 universal primer from                       USB, Cleveland, OH"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GTTTTCCCAGTCACGAC17                                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 ( A) LENGTH: 40 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..40                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB1888"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GTTCATGGATCCTTATTTGTCGTTAGGGTTATCAG GTATA40                                   (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..40                                                            (D) OTHER INFORMATION: /note="oligonucleotide BB1887"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TCAAGTGAATTCATGAAAAATTACTTATCTTTTGGGATGT40                                    (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1335 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..1335                                                         (D) OTHER INFORMATION: /note="Streptokinase gene from S.                      equisimilis"                                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 7..1326                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 7..1326                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GAATTCATGAAAAATTACTTATCTTTTGGGATGTTTGCACTGCTGTTT48                            MetLysAsnTyrLeuSerPheGlyMetPheAlaLeuLeuPhe                                    15 10                                                                         GCACTAACATTTGGAACAGTCAATTCTGTCCAAGCTATTGCTGGACCT96                            AlaLeuThrPheGlyThrValAsnSerValGlnAlaIleAlaGlyPro                              152025 30                                                                     GAGTGGCTGCTAGACCGTCCATCTGTCAACAACAGCCAATTAGTTGTT144                           GluTrpLeuLeuAspArgProSerValAsnAsnSerGlnLeuValVal                              3540 45                                                                       AGCGTTGCTGGTACTGTTGAGGGGACGAATCAAGACATTAGTCTTAAA192                           SerValAlaGlyThrValGluGlyThrAsnGlnAspIleSerLeuLys                              5055 60                                                                       TTTTTTGAAATTGACCTAACATCACGACCTGCTCATGGAGGAAAGACA240                           PhePheGluIleAspLeuThrSerArgProAlaHisGlyGlyLysThr                              65707 5                                                                       GAGCAAGGCTTAAGTCCAAAATCAAAACCATTTGCTACTGATAGTGGC288                           GluGlnGlyLeuSerProLysSerLysProPheAlaThrAspSerGly                              808590                                                                        GCGATG CCACATAAACTTGAAAAAGCTGACTTACTAAAGGCTATTCAA336                          AlaMetProHisLysLeuGluLysAlaAspLeuLeuLysAlaIleGln                              95100105110                                                                   GA ACAATTGATCGCTAACGTCCACAGTAACGACGACTACTTTGAGGTC384                          GluGlnLeuIleAlaAsnValHisSerAsnAspAspTyrPheGluVal                              115120125                                                                     A TTGATTTTGCAAGCGATGCAACCATTACTGATCGAAACGGCAAGGTC432                          IleAspPheAlaSerAspAlaThrIleThrAspArgAsnGlyLysVal                              130135140                                                                     TAC TTTGCTGACAAAGATGGTTCGGTAACCTTGCCGACCCAACCTGTC480                          TyrPheAlaAspLysAspGlySerValThrLeuProThrGlnProVal                              145150155                                                                     CAAGAATTT TTGCTAAGCGGACATGTGCGCGTTAGACCATATAAAGAA528                          GlnGluPheLeuLeuSerGlyHisValArgValArgProTyrLysGlu                              160165170                                                                     AAACCAATACAAAATCA AGCGAAATCTGTTGATGTGGAATATACTGTA576                          LysProIleGlnAsnGlnAlaLysSerValAspValGluTyrThrVal                              175180185190                                                                  CAGTTTACTCCCT TAAACCCTGATGACGATTTCAGACCAGGTCTCAAA624                          GlnPheThrProLeuAsnProAspAspAspPheArgProGlyLeuLys                              195200205                                                                     GATACTAAGCTA TTGAAAACACTAGCTATCGGTGACACCATCACATCT672                          AspThrLysLeuLeuLysThrLeuAlaIleGlyAspThrIleThrSer                              210215220                                                                     CAAGAATTACTAGCT CAAGCACAAAGCATTTTAAACAAAACCCATCCA720                          GlnGluLeuLeuAlaGlnAlaGlnSerIleLeuAsnLysThrHisPro                              225230235                                                                     GGCTATACGATTTATGAACG TGACTCCTCAATCGTCACTCATGACAAT768                          GlyTyrThrIleTyrGluArgAspSerSerIleValThrHisAspAsn                              240245250                                                                     GACATTTTCCGTACGATTTTACCAATGG ATCAAGAGTTTACTTACCAT816                          AspIlePheArgThrIleLeuProMetAspGlnGluPheThrTyrHis                              255260265270                                                                  GTCAAAAATCGGGAACAAGCTTAT GAGATCAATAAAAAATCTGGTCTG864                          ValLysAsnArgGluGlnAlaTyrGluIleAsnLysLysSerGlyLeu                              275280285                                                                     AATGAAGAAATAAACAACACTGAC CTGATCTCTGAGAAATATTACGTC912                          AsnGluGluIleAsnAsnThrAspLeuIleSerGluLysTyrTyrVal                              290295300                                                                     CTTAAAAAAGGGGAAAAGCCGTATGA TCCCTTTGATCGCAGTCACTTG960                          LeuLysLysGlyGluLysProTyrAspProPheAspArgSerHisLeu                              305310315                                                                     AAACTGTTCACCATCAAATACGTTGATGTCA ACACCAACGAATTGCTA1008                         LysLeuPheThrIleLysTyrValAspValAsnThrAsnGluLeuLeu                              320325330                                                                     AAAAGCGAGCAGCTCTTAACAGCTAGCGAACGTAACTTA GACTTCAGA1056                         LysSerGluGlnLeuLeuThrAlaSerGluArgAsnLeuAspPheArg                              335340345350                                                                  GATTTATACGATCCTCGTGATAAGGCTAAACTACTC TACAACAATCTC1104                         AspLeuTyrAspProArgAspLysAlaLysLeuLeuTyrAsnAsnLeu                              355360365                                                                     GATGCTTTTGGTATTATGGACTATACCTTAACTGG AAAAGTAGAAGAT1152                         AspAlaPheGlyIleMetAspTyrThrLeuThrGlyLysValGluAsp                              370375380                                                                     AATCACGATGACACCAACCGTATCATAACCGTTTAT ATGGGCAAGCGA1200                         AsnHisAspAspThrAsnArgIleIleThrValTyrMetGlyLysArg                              385390395                                                                     CCCGAAGGAGAGAATGCTAGCTATCATTTAGCCTATGATAAA GATCGT1248                         ProGluGlyGluAsnAlaSerTyrHisLeuAlaTyrAspLysAspArg                              400405410                                                                     TATACCGAAGAAGAACGAGAAGTTTACAGCTACCTGCGTTATACAGGG 1296                         TyrThrGluGluGluArgGluValTyrSerTyrLeuArgTyrThrGly                              415420425430                                                                  ACACCTATACCTGATAACCCTAACGACAAATAAGGATCC 1335                                  ThrProIleProAspAsnProAsnAspLys                                                435440                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 440 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      MetLysAsnTyrLeuSerPheGlyMetPheAlaLeuLeuPheAlaLeu                              151015                                                                        ThrPheGlyThrValAsnSerValGlnAlaIleAlaGlyProGluTrp                               202530                                                                       LeuLeuAspArgProSerValAsnAsnSerGlnLeuValValSerVal                              354045                                                                        AlaGlyThrValGluGlyTh rAsnGlnAspIleSerLeuLysPhePhe                             505560                                                                        GluIleAspLeuThrSerArgProAlaHisGlyGlyLysThrGluGln                              657075 80                                                                     GlyLeuSerProLysSerLysProPheAlaThrAspSerGlyAlaMet                              859095                                                                        ProHisLysLeuGluLysAlaAspLeuLeuLysAlaIleG lnGluGln                             100105110                                                                     LeuIleAlaAsnValHisSerAsnAspAspTyrPheGluValIleAsp                              115120125                                                                     PheAlaSer AspAlaThrIleThrAspArgAsnGlyLysValTyrPhe                             130135140                                                                     AlaAspLysAspGlySerValThrLeuProThrGlnProValGlnGlu                              145150 155160                                                                 PheLeuLeuSerGlyHisValArgValArgProTyrLysGluLysPro                              165170175                                                                     IleGlnAsnGlnAlaLysSerValAspVa lGluTyrThrValGlnPhe                             180185190                                                                     ThrProLeuAsnProAspAspAspPheArgProGlyLeuLysAspThr                              1952002 05                                                                    LysLeuLeuLysThrLeuAlaIleGlyAspThrIleThrSerGlnGlu                              210215220                                                                     LeuLeuAlaGlnAlaGlnSerIleLeuAsnLysThrHisProGlyTyr                              225 230235240                                                                 ThrIleTyrGluArgAspSerSerIleValThrHisAspAsnAspIle                              245250255                                                                     PheArgThrIleLeuPr oMetAspGlnGluPheThrTyrHisValLys                             260265270                                                                     AsnArgGluGlnAlaTyrGluIleAsnLysLysSerGlyLeuAsnGlu                              275280 285                                                                    GluIleAsnAsnThrAspLeuIleSerGluLysTyrTyrValLeuLys                              290295300                                                                     LysGlyGluLysProTyrAspProPheAspArgSerHisLeuLysLeu                               305310315320                                                                 PheThrIleLysTyrValAspValAsnThrAsnGluLeuLeuLysSer                              325330335                                                                     GluGln LeuLeuThrAlaSerGluArgAsnLeuAspPheArgAspLeu                             340345350                                                                     TyrAspProArgAspLysAlaLysLeuLeuTyrAsnAsnLeuAspAla                              355 360365                                                                    PheGlyIleMetAspTyrThrLeuThrGlyLysValGluAspAsnHis                              370375380                                                                     AspAspThrAsnArgIleIleThrValTyrMetGlyLy sArgProGlu                             385390395400                                                                  GlyGluAsnAlaSerTyrHisLeuAlaTyrAspLysAspArgTyrThr                              405410 415                                                                    GluGluGluArgGluValTyrSerTyrLeuArgTyrThrGlyThrPro                              420425430                                                                     IleProAspAsnProAsnAspLys                                                      435440                                                                        ( 2) INFORMATION FOR SEQ ID NO:16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..26                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2175"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GATAA GTAATTTTTCATATGAATTCG26                                                 (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                            (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2358"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CATGAGCAGGTCGTGATG18                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1317 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..1317                                                         (D) OTHER INFORMATION: /note="OmpAL fused to mature                           streptokinase gene"                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 4..1308                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                             (B) LOCATION: 4..1308                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CATATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTC48                            MetLysLysThrAlaIleAlaIleAlaValAlaLeuAlaGlyPhe                                 1 51015                                                                       GCGACCGTAGCGCAGGCCATTGCTGGACCTGAGTGGCTGCTAGACCGT96                            AlaThrValAlaGlnAlaIleAlaGlyProGluTrpLeuLeuAspArg                               202530                                                                       CCATCTGTCAACAACAGCCAATTAGTTGTTAGCGTTGCTGGTACTGTT144                           ProSerValAsnAsnSerGlnLeuValValSerValAlaGlyThrVal                               354045                                                                       GAGGGGACGAATCAAGACATTAGTCTTAAATTTTTTGAAATTGACCTA192                           GluGlyThrAsnGlnAspIleSerLeuLysPhePheGluIleAspLeu                               505560                                                                       ACATCACGACCTGCTCATGGAGGAAAGACAGAGCAAGGCTTAAGTCCA240                           ThrSerArgProAlaHisGlyGlyLysThrGluGlnGlyLeuSerPro                              65 7075                                                                       AAATCAAAACCATTTGCTACTGATAGTGGCGCGATGCCACATAAACTT288                           LysSerLysProPheAlaThrAspSerGlyAlaMetProHisLysLeu                              808 59095                                                                     GAAAAAGCTGACTTACTAAAGGCTATTCAAGAACAATTGATCGCTAAC336                           GluLysAlaAspLeuLeuLysAlaIleGlnGluGlnLeuIleAlaAsn                              1 00105110                                                                    GTCCACAGTAACGACGACTACTTTGAGGTCATTGATTTTGCAAGCGAT384                           ValHisSerAsnAspAspTyrPheGluValIleAspPheAlaSerAsp                              115 120125                                                                    GCAACCATTACTGATCGAAACGGCAAGGTCTACTTTGCTGACAAAGAT432                           AlaThrIleThrAspArgAsnGlyLysValTyrPheAlaAspLysAsp                              130 135140                                                                    GGTTCGGTAACCTTGCCGACCCAACCTGTCCAAGAATTTTTGCTAAGC480                           GlySerValThrLeuProThrGlnProValGlnGluPheLeuLeuSer                              14515 0155                                                                    GGACATGTGCGCGTTAGACCATATAAAGAAAAACCAATACAAAATCAA528                           GlyHisValArgValArgProTyrLysGluLysProIleGlnAsnGln                              160165 170175                                                                 GCGAAATCTGTTGATGTGGAATATACTGTACAGTTTACTCCCTTAAAC576                           AlaLysSerValAspValGluTyrThrValGlnPheThrProLeuAsn                              180 185190                                                                    CCTGATGACGATTTCAGACCAGGTCTCAAAGATACTAAGCTATTGAAA624                           ProAspAspAspPheArgProGlyLeuLysAspThrLysLeuLeuLys                              195 200205                                                                    ACACTAGCTATCGGTGACACCATCACATCTCAAGAATTACTAGCTCAA672                           ThrLeuAlaIleGlyAspThrIleThrSerGlnGluLeuLeuAlaGln                              210215 220                                                                    GCACAAAGCATTTTAAACAAAACCCATCCAGGCTATACGATTTATGAA720                           AlaGlnSerIleLeuAsnLysThrHisProGlyTyrThrIleTyrGlu                              225230 235                                                                    CGTGACTCCTCAATCGTCACTCATGACAATGACATTTTCCGTACGATT768                           ArgAspSerSerIleValThrHisAspAsnAspIlePheArgThrIle                              240245250 255                                                                 TTACCAATGGATCAAGAGTTTACTTACCATGTCAAAAATCGGGAACAA816                           LeuProMetAspGlnGluPheThrTyrHisValLysAsnArgGluGln                              260265 270                                                                    GCTTATGAGATCAATAAAAAATCTGGTCTGAATGAAGAAATAAACAAC864                           AlaTyrGluIleAsnLysLysSerGlyLeuAsnGluGluIleAsnAsn                              275280 285                                                                    ACTGACCTGATCTCTGAGAAATATTACGTCCTTAAAAAAGGGGAAAAG912                           ThrAspLeuIleSerGluLysTyrTyrValLeuLysLysGlyGluLys                              2902953 00                                                                    CCGTATGATCCCTTTGATCGCAGTCACTTGAAACTGTTCACCATCAAA960                           ProTyrAspProPheAspArgSerHisLeuLysLeuPheThrIleLys                              305310315                                                                     TACGT TGATGTCAACACCAACGAATTGCTAAAAAGCGAGCAGCTCTTA1008                         TyrValAspValAsnThrAsnGluLeuLeuLysSerGluGlnLeuLeu                              320325330335                                                                  A CAGCTAGCGAACGTAACTTAGACTTCAGAGATTTATACGATCCTCGT1056                         ThrAlaSerGluArgAsnLeuAspPheArgAspLeuTyrAspProArg                              340345350                                                                      GATAAGGCTAAACTACTCTACAACAATCTCGATGCTTTTGGTATTATG1104                         AspLysAlaLysLeuLeuTyrAsnAsnLeuAspAlaPheGlyIleMet                              355360365                                                                     GAC TATACCTTAACTGGAAAAGTAGAAGATAATCACGATGACACCAAC1152                         AspTyrThrLeuThrGlyLysValGluAspAsnHisAspAspThrAsn                              370375380                                                                     CGTATCAT AACCGTTTATATGGGCAAGCGACCCGAAGGAGAGAATGCT1200                         ArgIleIleThrValTyrMetGlyLysArgProGluGlyGluAsnAla                              385390395                                                                     AGCTATCATTTAGCCT ATGATAAAGATCGTTATACCGAAGAAGAACGA1248                         SerTyrHisLeuAlaTyrAspLysAspArgTyrThrGluGluGluArg                              400405410415                                                                  GAAGTTTACAGC TACCTGCGTTATACAGGGACACCTATACCTGATAAC1296                         GluValTyrSerTyrLeuArgTyrThrGlyThrProIleProAspAsn                              420425430                                                                     CCTAACGACAA ATAAGGATCC1317                                                    ProAsnAspLys                                                                  435                                                                           (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 435 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      MetLysLysThrAlaIleAlaIleAlaValAlaLeuAlaGlyPheAla                              151015                                                                        ThrValAlaGlnAlaIleAlaGlyProGluTrpLeuLeuAspArgPro                               202530                                                                       SerValAsnAsnSerGlnLeuValValSerValAlaGlyThrValGlu                              354045                                                                        GlyThrAsnGlnAspIleSer LeuLysPhePheGluIleAspLeuThr                             505560                                                                        SerArgProAlaHisGlyGlyLysThrGluGlnGlyLeuSerProLys                              657075 80                                                                     SerLysProPheAlaThrAspSerGlyAlaMetProHisLysLeuGlu                              859095                                                                        LysAlaAspLeuLeuLysAlaIleGlnGluGlnLeuIleAl aAsnVal                             100105110                                                                     HisSerAsnAspAspTyrPheGluValIleAspPheAlaSerAspAla                              115120125                                                                     ThrIleThr AspArgAsnGlyLysValTyrPheAlaAspLysAspGly                             130135140                                                                     SerValThrLeuProThrGlnProValGlnGluPheLeuLeuSerGly                              145150 155160                                                                 HisValArgValArgProTyrLysGluLysProIleGlnAsnGlnAla                              165170175                                                                     LysSerValAspValGluTyrThrValGln PheThrProLeuAsnPro                             180185190                                                                     AspAspAspPheArgProGlyLeuLysAspThrLysLeuLeuLysThr                              19520020 5                                                                    LeuAlaIleGlyAspThrIleThrSerGlnGluLeuLeuAlaGlnAla                              210215220                                                                     GlnSerIleLeuAsnLysThrHisProGlyTyrThrIleTyrGluArg                              225 230235240                                                                 AspSerSerIleValThrHisAspAsnAspIlePheArgThrIleLeu                              245250255                                                                     ProMetAspGlnGluPhe ThrTyrHisValLysAsnArgGluGlnAla                             260265270                                                                     TyrGluIleAsnLysLysSerGlyLeuAsnGluGluIleAsnAsnThr                              275280 285                                                                    AspLeuIleSerGluLysTyrTyrValLeuLysLysGlyGluLysPro                              290295300                                                                     TyrAspProPheAspArgSerHisLeuLysLeuPheThrIleLysTyr                              3 05310315320                                                                 ValAspValAsnThrAsnGluLeuLeuLysSerGluGlnLeuLeuThr                              325330335                                                                     AlaSer GluArgAsnLeuAspPheArgAspLeuTyrAspProArgAsp                             340345350                                                                     LysAlaLysLeuLeuTyrAsnAsnLeuAspAlaPheGlyIleMetAsp                              355 360365                                                                    TyrThrLeuThrGlyLysValGluAspAsnHisAspAspThrAsnArg                              370375380                                                                     IleIleThrValTyrMetGlyLysArgProGluGlyGlu AsnAlaSer                             385390395400                                                                  TyrHisLeuAlaTyrAspLysAspArgTyrThrGluGluGluArgGlu                              405410 415                                                                    ValTyrSerTyrLeuArgTyrThrGlyThrProIleProAspAsnPro                              420425430                                                                     AsnAspLys                                                                     435                                                                           (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..44                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB58"                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AGCTCGTAGACACTCTGCAGTTCGTTTGTGGTGACCGTGGCT TC44                               (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..30                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2658"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ACCGTAGCGCAGGCCATTGCTGGACCTGAG30                                              (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2753"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GACACCAACCGTATCAT17                                                           (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3510"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CACTATCAGTAGCAAAT 17                                                          (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..31                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3802"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GAAATACTTACATATGATTGCTGGACCTGAG31                                             (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1257 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..1257                                                         (D) OTHER INFORMATION: /note="Methionyl-streptokinase                         fusion protein"                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 4..1248                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 4..1248                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CATATGATTGCTGGACCTGAGTGGCTGCTAGACCGTCCATCTGTCAAC48                            MetIleAlaGlyProGluTrpLeuLeuAspArgProSerValAsn                                 15 1015                                                                       AACAGCCAATTAGTTGTTAGCGTTGCTGGTACTGTTGAGGGGACGAAT96                            AsnSerGlnLeuValValSerValAlaGlyThrValGluGlyThrAsn                              20 2530                                                                       CAAGACATTAGTCTTAAATTTTTTGAAATTGACCTAACATCACGACCT144                           GlnAspIleSerLeuLysPhePheGluIleAspLeuThrSerArgPro                              35 4045                                                                       GCTCATGGAGGAAAGACAGAGCAAGGCTTAAGTCCAAAATCAAAACCA192                           AlaHisGlyGlyLysThrGluGlnGlyLeuSerProLysSerLysPro                              5055 60                                                                       TTTGCTACTGATAGTGGCGCGATGCCACATAAACTTGAAAAAGCTGAC240                           PheAlaThrAspSerGlyAlaMetProHisLysLeuGluLysAlaAsp                              6570 75                                                                       TTACTAAAGGCTATTCAAGAACAATTGATCGCTAACGTCCACAGTAAC288                           LeuLeuLysAlaIleGlnGluGlnLeuIleAlaAsnValHisSerAsn                              808590 95                                                                     GACGACTACTTTGAGGTCATTGATTTTGCAAGCGATGCAACCATTACT336                           AspAspTyrPheGluValIleAspPheAlaSerAspAlaThrIleThr                              100105 110                                                                    GATCGAAACGGCAAGGTCTACTTTGCTGACAAAGATGGTTCGGTAACC384                           AspArgAsnGlyLysValTyrPheAlaAspLysAspGlySerValThr                              115120 125                                                                    TTGCCGACCCAACCTGTCCAAGAATTTTTGCTAAGCGGACATGTGCGC432                           LeuProThrGlnProValGlnGluPheLeuLeuSerGlyHisValArg                              130135 140                                                                    GTTAGACCATATAAAGAAAAACCAATACAAAATCAAGCGAAATCTGTT480                           ValArgProTyrLysGluLysProIleGlnAsnGlnAlaLysSerVal                              145150155                                                                     GAT GTGGAATATACTGTACAGTTTACTCCCTTAAACCCTGATGACGAT528                          AspValGluTyrThrValGlnPheThrProLeuAsnProAspAspAsp                              160165170175                                                                  TTCAGACCAGGTCTCAAAGATACTAAGCTATTGAAAACACTAGCTATC576                           PheArgProGlyLeuLysAspThrLysLeuLeuLysThrLeuAlaIle                              180185190                                                                     GGTGACACCATCACATCTCAAGAATTACTAGCTCAAGCACAAAGCATT624                           GlyAspThrIleThrSerGlnGluLeuLeuAlaGlnAlaGlnSerIle                              195200205                                                                      TTAAACAAAACCCATCCAGGCTATACGATTTATGAACGTGACTCCTCA672                          LeuAsnLysThrHisProGlyTyrThrIleTyrGluArgAspSerSer                              210215220                                                                     ATCGTC ACTCATGACAATGACATTTTCCGTACGATTTTACCAATGGAT720                          IleValThrHisAspAsnAspIlePheArgThrIleLeuProMetAsp                              225230235                                                                     CAAGAGTTTACTTA CCATGTCAAAAATCGGGAACAAGCTTATGAGATC768                          GlnGluPheThrTyrHisValLysAsnArgGluGlnAlaTyrGluIle                              240245250255                                                                  AATAAAAAAT CTGGTCTGAATGAAGAAATAAACAACACTGACCTGATC816                          AsnLysLysSerGlyLeuAsnGluGluIleAsnAsnThrAspLeuIle                              260265270                                                                     TCTGAGAAA TATTACGTCCTTAAAAAAGGGGAAAAGCCGTATGATCCC864                          SerGluLysTyrTyrValLeuLysLysGlyGluLysProTyrAspPro                              275280285                                                                     TTTGATCGCAG TCACTTGAAACTGTTCACCATCAAATACGTTGATGTC912                          PheAspArgSerHisLeuLysLeuPheThrIleLysTyrValAspVal                              290295300                                                                     AACACCAACGAATTGC TAAAAAGCGAGCAGCTCTTAACAGCTAGCGAA960                          AsnThrAsnGluLeuLeuLysSerGluGlnLeuLeuThrAlaSerGlu                              305310315                                                                     CGTAACTTAGACTTCAGAGATTTA TACGATCCTCGTGATAAGGCTAAA1008                         ArgAsnLeuAspPheArgAspLeuTyrAspProArgAspLysAlaLys                              320325330335                                                                  CTACTCTACAACAATCTCGAT GCTTTTGGTATTATGGACTATACCTTA1056                         LeuLeuTyrAsnAsnLeuAspAlaPheGlyIleMetAspTyrThrLeu                              340345350                                                                     ACTGGAAAAGTAGAAGATAA TCACGATGACACCAACCGTATCATAACC1104                         ThrGlyLysValGluAspAsnHisAspAspThrAsnArgIleIleThr                              355360365                                                                     GTTTATATGGGCAAGCGACCCG AAGGAGAGAATGCTAGCTATCATTTA1152                         ValTyrMetGlyLysArgProGluGlyGluAsnAlaSerTyrHisLeu                              370375380                                                                     GCCTATGATAAAGATCGTTATACCGAA GAAGAACGAGAAGTTTACAGC1200                         AlaTyrAspLysAspArgTyrThrGluGluGluArgGluValTyrSer                              385390395                                                                     TACCTGCGTTATACAGGGACACCTATACCTGATAAC CCTAACGACAAA1248                         TyrLeuArgTyrThrGlyThrProIleProAspAsnProAsnAspLys                              400405410415                                                                  TAAGGATCC 1257                                                                (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 415 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      MetIleAlaGlyProGluTrpLeuLeuAspArgProSerValAs nAsn                             151015                                                                        SerGlnLeuValValSerValAlaGlyThrValGluGlyThrAsnGln                              202530                                                                        AspIle SerLeuLysPhePheGluIleAspLeuThrSerArgProAla                             354045                                                                        HisGlyGlyLysThrGluGlnGlyLeuSerProLysSerLysProPhe                              50 5560                                                                       AlaThrAspSerGlyAlaMetProHisLysLeuGluLysAlaAspLeu                              65707580                                                                      LeuLysAlaIleGlnGluGlnLeuIleAlaAs nValHisSerAsnAsp                             859095                                                                        AspTyrPheGluValIleAspPheAlaSerAspAlaThrIleThrAsp                              100105 110                                                                    ArgAsnGlyLysValTyrPheAlaAspLysAspGlySerValThrLeu                              115120125                                                                     ProThrGlnProValGlnGluPheLeuLeuSerGlyHisValArgVal                              130 135140                                                                    ArgProTyrLysGluLysProIleGlnAsnGlnAlaLysSerValAsp                              145150155160                                                                  ValGluTyrThrValGlnPhe ThrProLeuAsnProAspAspAspPhe                             165170175                                                                     ArgProGlyLeuLysAspThrLysLeuLeuLysThrLeuAlaIleGly                              180185 190                                                                    AspThrIleThrSerGlnGluLeuLeuAlaGlnAlaGlnSerIleLeu                              195200205                                                                     AsnLysThrHisProGlyTyrThrIleTyrGluArgAspSerSe rIle                             210215220                                                                     ValThrHisAspAsnAspIlePheArgThrIleLeuProMetAspGln                              225230235240                                                                  GluPheThr TyrHisValLysAsnArgGluGlnAlaTyrGluIleAsn                             245250255                                                                     LysLysSerGlyLeuAsnGluGluIleAsnAsnThrAspLeuIleSer                              260 265270                                                                    GluLysTyrTyrValLeuLysLysGlyGluLysProTyrAspProPhe                              275280285                                                                     AspArgSerHisLeuLysLeuPheThrIleLys TyrValAspValAsn                             290295300                                                                     ThrAsnGluLeuLeuLysSerGluGlnLeuLeuThrAlaSerGluArg                              305310315320                                                                  AsnLeuAspPheArgAspLeuTyrAspProArgAspLysAlaLysLeu                              325330335                                                                     LeuTyrAsnAsnLeuAspAlaPheGlyIleMetAspTyrThrLeuThr                               340345350                                                                    GlyLysValGluAspAsnHisAspAspThrAsnArgIleIleThrVal                              355360365                                                                     TyrMetGlyLysArgProGlu GlyGluAsnAlaSerTyrHisLeuAla                             370375380                                                                     TyrAspLysAspArgTyrThrGluGluGluArgGluValTyrSerTyr                              385390395 400                                                                 LeuArgTyrThrGlyThrProIleProAspAsnProAsnAspLys                                 405410415                                                                     (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1512 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..1512                                                         (D) OTHER INFORMATION: /note="Streptokinase                                   fused to a yeast alpha-factor"                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 7..1503                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                             (B) LOCATION: 7..1503                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AGATCTATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCA48                            MetArgPheProSerIlePheThrAlaValLeuPheAlaAla                                     1510                                                                         TCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACG96                            SerSerAlaLeuAlaAlaProValAsnThrThrThrGluAspGluThr                              15 202530                                                                     GCACAAATTCCGGCTGAAGCTGTCATCGGTTACTTAGATTTAGAAGGG144                           AlaGlnIleProAlaGluAlaValIleGlyTyrLeuAspLeuGluGly                               354045                                                                       GATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGG192                           AspPheAspValAlaValLeuProPheSerAsnSerThrAsnAsnGly                              5 05560                                                                       TTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAA240                           LeuLeuPheIleAsnThrThrIleAlaSerIleAlaAlaLysGluGlu                              65 7075                                                                       GGGGTAAGCTTGGATAAAAGAATTGCTGGACCTGAGTGGCTGCTAGAC288                           GlyValSerLeuAspLysArgIleAlaGlyProGluTrpLeuLeuAsp                              80 8590                                                                       CGTCCATCTGTCAACAACAGCCAATTAGTTGTTAGCGTTGCTGGTACT336                           ArgProSerValAsnAsnSerGlnLeuValValSerValAlaGlyThr                              95100 105110                                                                  GTTGAGGGGACGAATCAAGACATTAGTCTTAAATTTTTTGAAATTGAC384                           ValGluGlyThrAsnGlnAspIleSerLeuLysPhePheGluIleAsp                              115 120125                                                                    CTAACATCACGACCTGCTCATGGAGGAAAGACAGAGCAAGGCTTAAGT432                           LeuThrSerArgProAlaHisGlyGlyLysThrGluGlnGlyLeuSer                              130 135140                                                                    CCAAAATCAAAACCATTTGCTACTGATAGTGGCGCGATGCCACATAAA480                           ProLysSerLysProPheAlaThrAspSerGlyAlaMetProHisLys                              145150 155                                                                    CTTGAAAAAGCTGACTTACTAAAGGCTATTCAAGAACAATTGATCGCT528                           LeuGluLysAlaAspLeuLeuLysAlaIleGlnGluGlnLeuIleAla                              160165 170                                                                    AACGTCCACAGTAACGACGACTACTTTGAGGTCATTGATTTTGCAAGC576                           AsnValHisSerAsnAspAspTyrPheGluValIleAspPheAlaSer                              175180185 190                                                                 GATGCAACCATTACTGATCGAAACGGCAAGGTCTACTTTGCTGACAAA624                           AspAlaThrIleThrAspArgAsnGlyLysValTyrPheAlaAspLys                              195200 205                                                                    GATGGTTCGGTAACCTTGCCGACCCAACCTGTCCAAGAATTTTTGCTA672                           AspGlySerValThrLeuProThrGlnProValGlnGluPheLeuLeu                              210215 220                                                                    AGCGGACATGTGCGCGTTAGACCATATAAAGAAAAACCAATACAAAAT720                           SerGlyHisValArgValArgProTyrLysGluLysProIleGlnAsn                              225230 235                                                                    CAAGCGAAATCTGTTGATGTGGAATATACTGTACAGTTTACTCCCTTA768                           GlnAlaLysSerValAspValGluTyrThrValGlnPheThrProLeu                              240245250                                                                     AAC CCTGATGACGATTTCAGACCAGGTCTCAAAGATACTAAGCTATTG816                          AsnProAspAspAspPheArgProGlyLeuLysAspThrLysLeuLeu                              255260265270                                                                  AAAACACTAGCTATCGGTGACACCATCACATCTCAAGAATTACTAGCT864                           LysThrLeuAlaIleGlyAspThrIleThrSerGlnGluLeuLeuAla                              275280285                                                                     CAAGCACAAAGCATTTTAAACAAAACCCATCCAGGCTATACGATTTAT912                           GlnAlaGlnSerIleLeuAsnLysThrHisProGlyTyrThrIleTyr                              290295300                                                                      GAACGTGACTCCTCAATCGTCACTCATGACAATGACATTTTCCGTACG960                          GluArgAspSerSerIleValThrHisAspAsnAspIlePheArgThr                              305310315                                                                     ATTTTA CCAATGGATCAAGAGTTTACTTACCATGTCAAAAATCGGGAA1008                         IleLeuProMetAspGlnGluPheThrTyrHisValLysAsnArgGlu                              320325330                                                                     CAAGCTTATGAGAT CAATAAAAAATCTGGTCTGAATGAAGAAATAAAC1056                         GlnAlaTyrGluIleAsnLysLysSerGlyLeuAsnGluGluIleAsn                              335340345350                                                                  AACACTGACC TGATCTCTGAGAAATATTACGTCCTTAAAAAAGGGGAA1104                         AsnThrAspLeuIleSerGluLysTyrTyrValLeuLysLysGlyGlu                              355360365                                                                     AAGCCGTAT GATCCCTTTGATCGCAGTCACTTGAAACTGTTCACCATC1152                         LysProTyrAspProPheAspArgSerHisLeuLysLeuPheThrIle                              370375380                                                                     AAATACGTTGAT GTCAACACCAACGAATTGCTAAAAAGCGAGCAGCTC1200                         LysTyrValAspValAsnThrAsnGluLeuLeuLysSerGluGlnLeu                              385390395                                                                     TTAACAGCTAGCGAACG TAACTTAGACTTCAGAGATTTATACGATCCT1248                         LeuThrAlaSerGluArgAsnLeuAspPheArgAspLeuTyrAspPro                              400405410                                                                     CGTGATAAGGCTAAACTACTCTACA ACAATCTCGATGCTTTTGGTATT1296                         ArgAspLysAlaLysLeuLeuTyrAsnAsnLeuAspAlaPheGlyIle                              415420425430                                                                  ATGGACTATACCTTAACTGGA AAAGTAGAAGATAATCACGATGACACC1344                         MetAspTyrThrLeuThrGlyLysValGluAspAsnHisAspAspThr                              435440445                                                                     AACCGTATCATAACCGTTTAT ATGGGCAAGCGACCCGAAGGAGAGAAT1392                         AsnArgIleIleThrValTyrMetGlyLysArgProGluGlyGluAsn                              450455460                                                                     GCTAGCTATCATTTAGCCTATGA TAAAGATCGTTATACCGAAGAAGAA1440                         AlaSerTyrHisLeuAlaTyrAspLysAspArgTyrThrGluGluGlu                              465470475                                                                     CGAGAAGTTTACAGCTACCTGCGTTATA CAGGGACACCTATACCTGAT1488                         ArgGluValTyrSerTyrLeuArgTyrThrGlyThrProIleProAsp                              480485490                                                                     AACCCTAACGACAAATAAGGATCC 1512                                                 AsnProAsnAspLys                                                               495                                                                           (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 499 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      MetArgPheProSerIlePheThrAlaVa lLeuPheAlaAlaSerSer                             151015                                                                        AlaLeuAlaAlaProValAsnThrThrThrGluAspGluThrAlaGln                              2025 30                                                                       IleProAlaGluAlaValIleGlyTyrLeuAspLeuGluGlyAspPhe                              354045                                                                        AspValAlaValLeuProPheSerAsnSerThrAsnAsnGlyLeuLeu                               505560                                                                       PheIleAsnThrThrIleAlaSerIleAlaAlaLysGluGluGlyVal                              65707580                                                                      SerLeuAspLysArgIle AlaGlyProGluTrpLeuLeuAspArgPro                             859095                                                                        SerValAsnAsnSerGlnLeuValValSerValAlaGlyThrValGlu                              100 105110                                                                    GlyThrAsnGlnAspIleSerLeuLysPhePheGluIleAspLeuThr                              115120125                                                                     SerArgProAlaHisGlyGlyLysThrGluGlnGlyLeuSe rProLys                             130135140                                                                     SerLysProPheAlaThrAspSerGlyAlaMetProHisLysLeuGlu                              145150155160                                                                  LysAla AspLeuLeuLysAlaIleGlnGluGlnLeuIleAlaAsnVal                             165170175                                                                     HisSerAsnAspAspTyrPheGluValIleAspPheAlaSerAspAla                              180 185190                                                                    ThrIleThrAspArgAsnGlyLysValTyrPheAlaAspLysAspGly                              195200205                                                                     SerValThrLeuProThrGlnProValGln GluPheLeuLeuSerGly                             210215220                                                                     HisValArgValArgProTyrLysGluLysProIleGlnAsnGlnAla                              225230235 240                                                                 LysSerValAspValGluTyrThrValGlnPheThrProLeuAsnPro                              245250255                                                                     AspAspAspPheArgProGlyLeuLysAspThrLysLeuLeuLysThr                               260265270                                                                    LeuAlaIleGlyAspThrIleThrSerGlnGluLeuLeuAlaGlnAla                              275280285                                                                     GlnSerIleLeuAsnLys ThrHisProGlyTyrThrIleTyrGluArg                             290295300                                                                     AspSerSerIleValThrHisAspAsnAspIlePheArgThrIleLeu                              305310315 320                                                                 ProMetAspGlnGluPheThrTyrHisValLysAsnArgGluGlnAla                              325330335                                                                     TyrGluIleAsnLysLysSerGlyLeuAsnGluGluIle AsnAsnThr                             340345350                                                                     AspLeuIleSerGluLysTyrTyrValLeuLysLysGlyGluLysPro                              355360365                                                                     TyrAspP roPheAspArgSerHisLeuLysLeuPheThrIleLysTyr                             370375380                                                                     ValAspValAsnThrAsnGluLeuLeuLysSerGluGlnLeuLeuThr                              385390 395400                                                                 AlaSerGluArgAsnLeuAspPheArgAspLeuTyrAspProArgAsp                              405410415                                                                     LysAlaLysLeuLeuTyrAsnAsnLeu AspAlaPheGlyIleMetAsp                             420425430                                                                     TyrThrLeuThrGlyLysValGluAspAsnHisAspAspThrAsnArg                              435440 445                                                                    IleIleThrValTyrMetGlyLysArgProGluGlyGluAsnAlaSer                              450455460                                                                     TyrHisLeuAlaTyrAspLysAspArgTyrThrGluGluGluArgGlu                              465 470475480                                                                 ValTyrSerTyrLeuArgTyrThrGlyThrProIleProAspAsnPro                              485490495                                                                     AsnAspLys                                                                     (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..36                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3624"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GTCCAAGCTA AGCTTGGATAAAAGAATTGCTGGACC36                                       (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1119 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..1119                                                         (D) OTHER INFORMATION: /note="Truncated Met-streptokinase                     (aa16-383)"                                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 4..1110                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 4..1110                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CATATGAGCCAA TTAGTTGTTAGCGTTGCTGGTACTGTTGAGGGGACG48                           MetSerGlnLeuValValSerValAlaGlyThrValGluGlyThr                                 151015                                                                        AATCAAGA CATTAGTCTTAAATTTTTTGAAATTGACCTAACATCACGA96                           AsnGlnAspIleSerLeuLysPhePheGluIleAspLeuThrSerArg                              202530                                                                        CCTGCTC ATGGAGGAAAGACAGAGCAAGGCTTAAGTCCAAAATCAAAA144                          ProAlaHisGlyGlyLysThrGluGlnGlyLeuSerProLysSerLys                              354045                                                                        CCATTTGCT ACTGATAGTGGCGCGATGCCACATAAACTTGAAAAAGCT192                          ProPheAlaThrAspSerGlyAlaMetProHisLysLeuGluLysAla                              505560                                                                        GACTTACTAAAGGCT ATTCAAGAACAATTGATCGCTAACGTCCACAGT240                          AspLeuLeuLysAlaIleGlnGluGlnLeuIleAlaAsnValHisSer                              657075                                                                        AACGACGACTACTTTGAGGTCAT TGATTTTGCAAGCGATGCAACCATT288                          AsnAspAspTyrPheGluValIleAspPheAlaSerAspAlaThrIle                              80859095                                                                      ACTGATCGAAACGGCAAGG TCTACTTTGCTGACAAAGATGGTTCGGTA336                          ThrAspArgAsnGlyLysValTyrPheAlaAspLysAspGlySerVal                              100105110                                                                     ACCTTGCCGACCCAACCT GTCCAAGAATTTTTGCTAAGCGGACATGTG384                          ThrLeuProThrGlnProValGlnGluPheLeuLeuSerGlyHisVal                              115120125                                                                     CGCGTTAGACCATATAAAGAA AAACCAATACAAAATCAAGCGAAATCT432                          ArgValArgProTyrLysGluLysProIleGlnAsnGlnAlaLysSer                              130135140                                                                     GTTGATGTGGAATATACTGTACAGTT TACTCCCTTAAACCCTGATGAC480                          ValAspValGluTyrThrValGlnPheThrProLeuAsnProAspAsp                              145150155                                                                     GATTTCAGACCAGGTCTCAAAGATACTAAGCTAT TGAAAACACTAGCT528                          AspPheArgProGlyLeuLysAspThrLysLeuLeuLysThrLeuAla                              160165170175                                                                  ATCGGTGACACCATCACATCTCAAGAATTA CTAGCTCAAGCACAAAGC576                          IleGlyAspThrIleThrSerGlnGluLeuLeuAlaGlnAlaGlnSer                              180185190                                                                     ATTTTAAACAAAACCCATCCAGGCTATACG ATTTATGAACGTGACTCC624                          IleLeuAsnLysThrHisProGlyTyrThrIleTyrGluArgAspSer                              195200205                                                                     TCAATCGTCACTCATGACAATGACATTTTCCG TACGATTTTACCAATG672                          SerIleValThrHisAspAsnAspIlePheArgThrIleLeuProMet                              210215220                                                                     GATCAAGAGTTTACTTACCATGTCAAAAATCGGGAAC AAGCTTATGAG720                          AspGlnGluPheThrTyrHisValLysAsnArgGluGlnAlaTyrGlu                              225230235                                                                     ATCAATAAAAAATCTGGTCTGAATGAAGAAATAAACAACACTGAC CTG768                          IleAsnLysLysSerGlyLeuAsnGluGluIleAsnAsnThrAspLeu                              240245250255                                                                  ATCTCTGAGAAATATTACGTCCTTAAAAAAGGGGAAAAGCCG TATGAT816                          IleSerGluLysTyrTyrValLeuLysLysGlyGluLysProTyrAsp                              260265270                                                                     CCCTTTGATCGCAGTCACTTGAAACTGTTCACCATCAAATA CGTTGAT864                          ProPheAspArgSerHisLeuLysLeuPheThrIleLysTyrValAsp                              275280285                                                                     GTCAACACCAACGAATTGCTAAAAAGCGAGCAGCTCTTAACAG CTAGC912                          ValAsnThrAsnGluLeuLeuLysSerGluGlnLeuLeuThrAlaSer                              290295300                                                                     GAACGTAACTTAGACTTCAGAGATTTATACGATCCTCGTGATAAGGCT 960                          GluArgAsnLeuAspPheArgAspLeuTyrAspProArgAspLysAla                              305310315                                                                     AAACTACTCTACAACAATCTCGATGCTTTTGGTATTATGGACTATACC1008                           LysLeuLeuTyrAsnAsnLeuAspAlaPheGlyIleMetAspTyrThr                             320325330335                                                                  TTAACTGGAAAAGTAGAAGATAATCACGATGACACCAACCGTATCATA1 056                         LeuThrGlyLysValGluAspAsnHisAspAspThrAsnArgIleIle                              340345350                                                                     ACCGTTTATATGGGCAAGCGACCCGAAGGAGAGAATGCTAGCTATCAT 1104                         ThrValTyrMetGlyLysArgProGluGlyGluAsnAlaSerTyrHis                              355360365                                                                     TTAGCCTAAGGATCC111 9                                                          LeuAla                                                                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 369 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      MetSerGlnLeuValValSerValAlaGlyThrValGluGlyThrAsn                              1 51015                                                                       GlnAspIleSerLeuLysPhePheGluIleAspLeuThrSerArgPro                              202530                                                                        AlaHisGlyGlyLysThrGlu GlnGlyLeuSerProLysSerLysPro                             354045                                                                        PheAlaThrAspSerGlyAlaMetProHisLysLeuGluLysAlaAsp                              5055 60                                                                       LeuLeuLysAlaIleGlnGluGlnLeuIleAlaAsnValHisSerAsn                              65707580                                                                      AspAspTyrPheGluValIleAspPheAlaSerAspAlaThrIleThr                              859095                                                                        AspArgAsnGlyLysValTyrPheAlaAspLysAspGlySerValThr                              100105110                                                                     LeuProThrG lnProValGlnGluPheLeuLeuSerGlyHisValArg                             115120125                                                                     ValArgProTyrLysGluLysProIleGlnAsnGlnAlaLysSerVal                              130135 140                                                                    AspValGluTyrThrValGlnPheThrProLeuAsnProAspAspAsp                              145150155160                                                                  PheArgProGlyLeuLysAspThrLysLeuLeuLys ThrLeuAlaIle                             165170175                                                                     GlyAspThrIleThrSerGlnGluLeuLeuAlaGlnAlaGlnSerIle                              180185190                                                                     LeuAsnLysThrHisProGlyTyrThrIleTyrGluArgAspSerSer                              195200205                                                                     IleValThrHisAspAsnAspIlePheArgThrIleLeuProMetAsp                              210 215220                                                                    GlnGluPheThrTyrHisValLysAsnArgGluGlnAlaTyrGluIle                              225230235240                                                                  AsnLysLysSerGlyLeuAsnGlu GluIleAsnAsnThrAspLeuIle                             245250255                                                                     SerGluLysTyrTyrValLeuLysLysGlyGluLysProTyrAspPro                              260265 270                                                                    PheAspArgSerHisLeuLysLeuPheThrIleLysTyrValAspVal                              275280285                                                                     AsnThrAsnGluLeuLeuLysSerGluGlnLeuLeuThrAlaSerGlu                              290295300                                                                     ArgAsnLeuAspPheArgAspLeuTyrAspProArgAspLysAlaLys                              305310315320                                                                  LeuLeuTyrAsnA snLeuAspAlaPheGlyIleMetAspTyrThrLeu                             325330335                                                                     ThrGlyLysValGluAspAsnHisAspAspThrAsnArgIleIleThr                              340 345350                                                                    ValTyrMetGlyLysArgProGluGlyGluAsnAlaSerTyrHisLeu                              355360365                                                                     Ala                                                                           (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..33                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3862"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GAAATACTTACATATGAGCCAATTAGTTGTTAG 33                                          (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..28                                                           (D ) OTHER INFORMATION: /note="oligonucleotide BB3904"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CCCGGGGATCCTTAGGCTAAATGATAGC28                                                (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2589 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..2589                                                         (D) OTHER INFORMATION: /note=                                                 "OmpAL-Streptokinase-streptokinase fusion linked                              by thrombin- cleavable VELQGVVPRG"                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 4..2580                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 4..2580                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CATATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTC48                            MetLysLysThrAlaIleAlaIleAlaValAlaLeuAlaGlyPhe                                 15 1015                                                                       GCGACCGTAGCGCAGGCCATTGCTGGACCTGAGTGGCTGCTAGACCGT96                            AlaThrValAlaGlnAlaIleAlaGlyProGluTrpLeuLeuAspArg                              20 2530                                                                       CCATCTGTCAACAACAGCCAATTAGTTGTTAGCGTTGCTGGTACTGTT144                           ProSerValAsnAsnSerGlnLeuValValSerValAlaGlyThrVal                              35 4045                                                                       GAGGGGACGAATCAAGACATTAGTCTTAAATTTTTTGAAATTGACCTA192                           GluGlyThrAsnGlnAspIleSerLeuLysPhePheGluIleAspLeu                              50 5560                                                                       ACATCACGACCTGCTCATGGAGGAAAGACAGAGCAAGGCTTAAGTCCA240                           ThrSerArgProAlaHisGlyGlyLysThrGluGlnGlyLeuSerPro                              6570 75                                                                       AAATCAAAACCATTTGCTACTGATAGTGGCGCGATGCCACATAAACTT288                           LysSerLysProPheAlaThrAspSerGlyAlaMetProHisLysLeu                              8085 9095                                                                     GAAAAAGCTGACTTACTAAAGGCTATTCAAGAACAATTGATCGCTAAC336                           GluLysAlaAspLeuLeuLysAlaIleGlnGluGlnLeuIleAlaAsn                              100 105110                                                                    GTCCACAGTAACGACGACTACTTTGAGGTCATTGATTTTGCAAGCGAT384                           ValHisSerAsnAspAspTyrPheGluValIleAspPheAlaSerAsp                              115120 125                                                                    GCAACCATTACTGATCGAAACGGCAAGGTCTACTTTGCTGACAAAGAT432                           AlaThrIleThrAspArgAsnGlyLysValTyrPheAlaAspLysAsp                              130135 140                                                                    GGTTCGGTAACCTTGCCGACCCAACCTGTCCAAGAATTTTTGCTAAGC480                           GlySerValThrLeuProThrGlnProValGlnGluPheLeuLeuSer                              1451501 55                                                                    GGACATGTGCGCGTTAGACCATATAAAGAAAAACCAATACAAAATCAA528                           GlyHisValArgValArgProTyrLysGluLysProIleGlnAsnGln                              160165170 175                                                                 GCGAAATCTGTTGATGTGGAATATACTGTACAGTTTACTCCCTTAAAC576                           AlaLysSerValAspValGluTyrThrValGlnPheThrProLeuAsn                              180185 190                                                                    CCTGATGACGATTTCAGACCAGGTCTCAAAGATACTAAGCTATTGAAA624                           ProAspAspAspPheArgProGlyLeuLysAspThrLysLeuLeuLys                              195200 205                                                                    ACACTAGCTATCGGTGACACCATCACATCTCAAGAATTACTAGCTCAA672                           ThrLeuAlaIleGlyAspThrIleThrSerGlnGluLeuLeuAlaGln                              210215220                                                                      GCACAAAGCATTTTAAACAAAACCCATCCAGGCTATACGATTTATGAA720                          AlaGlnSerIleLeuAsnLysThrHisProGlyTyrThrIleTyrGlu                              225230235                                                                     CGTGACTC CTCAATCGTCACTCATGACAATGACATTTTCCGTACGATT768                          ArgAspSerSerIleValThrHisAspAsnAspIlePheArgThrIle                              240245250255                                                                  TTAC CAATGGATCAAGAGTTTACTTACCATGTCAAAAATCGGGAACAA816                          LeuProMetAspGlnGluPheThrTyrHisValLysAsnArgGluGln                              260265270                                                                     GCT TATGAGATCAATAAAAAATCTGGTCTGAATGAAGAAATAAACAAC864                          AlaTyrGluIleAsnLysLysSerGlyLeuAsnGluGluIleAsnAsn                              275280285                                                                     ACTGA CCTGATCTCTGAGAAATATTACGTCCTTAAAAAAGGGGAAAAG912                          ThrAspLeuIleSerGluLysTyrTyrValLeuLysLysGlyGluLys                              290295300                                                                     CCGTATGATC CCTTTGATCGCAGTCACTTGAAACTGTTCACCATCAAA960                          ProTyrAspProPheAspArgSerHisLeuLysLeuPheThrIleLys                              305310315                                                                     TACGTTGATGTCAACACC AACGAATTGCTAAAAAGCGAGCAGCTCTTA1008                         TyrValAspValAsnThrAsnGluLeuLeuLysSerGluGlnLeuLeu                              320325330335                                                                  ACAGCTAGCGAACGT AACTTAGACTTCAGAGATTTATACGATCCTCGT1056                         ThrAlaSerGluArgAsnLeuAspPheArgAspLeuTyrAspProArg                              340345350                                                                     GATAAGGCTAAACT ACTCTACAACAATCTCGATGCTTTTGGTATTATG1104                         AspLysAlaLysLeuLeuTyrAsnAsnLeuAspAlaPheGlyIleMet                              355360365                                                                     GACTATACCTTAACTG GAAAAGTAGAAGATAATCACGATGACACCAAC1152                         AspTyrThrLeuThrGlyLysValGluAspAsnHisAspAspThrAsn                              370375380                                                                     CGTATCATAACCGTTTATATG GGCAAGCGACCCGAAGGAGAGAATGCT1200                         ArgIleIleThrValTyrMetGlyLysArgProGluGlyGluAsnAla                              385390395                                                                     AGCTATCATTTAGCCTATGATAAAGATCGT TATACCGAAGAAGAACGA1248                         SerTyrHisLeuAlaTyrAspLysAspArgTyrThrGluGluGluArg                              400405410415                                                                  GAAGTTTACAGCTACCTGCGTTATAC AGGGACACCTATACCTGATAAC1296                         GluValTyrSerTyrLeuArgTyrThrGlyThrProIleProAspAsn                              420425430                                                                     CCTAACGACAAAGTAGAGCTGCAGG GAGTAGTTCCTCGTGGAATTGCT1344                         ProAsnAspLysValGluLeuGlnGlyValValProArgGlyIleAla                              435440445                                                                     GGACCTGAGTGGCTGCTAGACCGTCCA TCTGTCAACAACAGCCAATTA1392                         GlyProGluTrpLeuLeuAspArgProSerValAsnAsnSerGlnLeu                              450455460                                                                     GTTGTTAGCGTTGCTGGTACTGTTGAGGGGACG AATCAAGACATTAGT1440                         ValValSerValAlaGlyThrValGluGlyThrAsnGlnAspIleSer                              465470475                                                                     CTTAAATTTTTTGAAATTGACCTAACATCACGACCTGCTCA TGGAGGA1488                         LeuLysPhePheGluIleAspLeuThrSerArgProAlaHisGlyGly                              480485490495                                                                  AAGACAGAGCAAGGCTTAAGTCCAAAATCAAAACCAT TTGCTACTGAT1536                         LysThrGluGlnGlyLeuSerProLysSerLysProPheAlaThrAsp                              500505510                                                                     AGTGGCGCGATGCCACATAAACTTGAAAAAGCTGAC TTACTAAAGGCT1584                         SerGlyAlaMetProHisLysLeuGluLysAlaAspLeuLeuLysAla                              515520525                                                                     ATTCAAGAACAATTGATCGCTAACGTCCACAGTAACGA CGACTACTTT1632                         IleGlnGluGlnLeuIleAlaAsnValHisSerAsnAspAspTyrPhe                              530535540                                                                     GAGGTCATTGATTTTGCAAGCGATGCAACCATTACTGATCGAA ACGGC1680                         GluValIleAspPheAlaSerAspAlaThrIleThrAspArgAsnGly                              545550555                                                                     AAGGTCTACTTTGCTGACAAAGATGGTTCGGTAACCTTGCCGACCCAA 1728                         LysValTyrPheAlaAspLysAspGlySerValThrLeuProThrGln                              560565570575                                                                  CCTGTCCAAGAATTTTTGCTAAGCGGACATGTGCGCGTTAGACCATAT 1776                         ProValGlnGluPheLeuLeuSerGlyHisValArgValArgProTyr                              580585590                                                                     AAAGAAAAACCAATACAAAATCAAGCGAAATCTGTTGATGTGGAATA T1824                         LysGluLysProIleGlnAsnGlnAlaLysSerValAspValGluTyr                              595600605                                                                     ACTGTACAGTTTACTCCCTTAAACCCTGATGACGATTTCAGACCAGGT 1872                         ThrValGlnPheThrProLeuAsnProAspAspAspPheArgProGly                              610615620                                                                     CTCAAAGATACTAAGCTATTGAAAACACTAGCTATCGGTGACACCATC1920                          LeuLysAspThrLysLeuLeuLysThrLeuAlaIleGlyAspThrIle                              625630635                                                                     ACATCTCAAGAATTACTAGCTCAAGCACAAAGCATTTTAAACAAAACC1968                          ThrSerG lnGluLeuLeuAlaGlnAlaGlnSerIleLeuAsnLysThr                             640645650655                                                                  CATCCAGGCTATACGATTTATGAACGTGACTCCTCAATCGTCACTCAT2016                          His ProGlyTyrThrIleTyrGluArgAspSerSerIleValThrHis                             660665670                                                                     GACAATGACATTTTCCGTACGATTTTACCAATGGATCAAGAGTTTACT2064                          Asp AsnAspIlePheArgThrIleLeuProMetAspGlnGluPheThr                             675680685                                                                     TACCATGTCAAAAATCGGGAACAAGCTTATGAGATCAATAAAAAATCT2112                          TyrHi sValLysAsnArgGluGlnAlaTyrGluIleAsnLysLysSer                             690695700                                                                     GGTCTGAATGAAGAAATAAACAACACTGACCTGATCTCTGAGAAATAT2160                          GlyLeuAsnG luGluIleAsnAsnThrAspLeuIleSerGluLysTyr                             705710715                                                                     TACGTCCTTAAAAAAGGGGAAAAGCCGTATGATCCCTTTGATCGCAGT2208                          TyrValLeuLysLysGly GluLysProTyrAspProPheAspArgSer                             720725730735                                                                  CACTTGAAACTGTTCACCATCAAATACGTTGATGTCAACACCAACGAA2256                          HisLeuLysLeuPh eThrIleLysTyrValAspValAsnThrAsnGlu                             740745750                                                                     TTGCTAAAAAGCGAGCAGCTCTTAACAGCTAGCGAACGTAACTTAGAC2304                          LeuLeuLysSerG luGlnLeuLeuThrAlaSerGluArgAsnLeuAsp                             755760765                                                                     TTCAGAGATTTATACGATCCTCGTGATAAGGCTAAACTACTCTACAAC2352                          PheArgAspLeuTyr AspProArgAspLysAlaLysLeuLeuTyrAsn                             770775780                                                                     AATCTCGATGCTTTTGGTATTATGGACTATACCTTAACTGGAAAAGTA2400                          AsnLeuAspAlaPheGlyIle MetAspTyrThrLeuThrGlyLysVal                             785790795                                                                     GAAGATAATCACGATGACACCAACCGTATCATAACCGTTTATATGGGC2448                          GluAspAsnHisAspAspThrAsnArgIl eIleThrValTyrMetGly                             800805810815                                                                  AAGCGACCCGAAGGAGAGAATGCTAGCTATCATTTAGCCTATGATAAA2496                          LysArgProGluGlyGluAsnAlaS erTyrHisLeuAlaTyrAspLys                             820825830                                                                     GATCGTTATACCGAAGAAGAACGAGAAGTTTACAGCTACCTGCGTTAT2544                          AspArgTyrThrGluGluGluArg GluValTyrSerTyrLeuArgTyr                             835840845                                                                     ACAGGGACACCTATACCTGATAACCCTAACGACAAATAAGGATCC2589                             ThrGlyThrProIleProAspAsnPro AsnAspLys                                         850855                                                                        (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 859 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      MetLysLysThrAlaIleAlaIleAlaValAla LeuAlaGlyPheAla                             151015                                                                        ThrValAlaGlnAlaIleAlaGlyProGluTrpLeuLeuAspArgPro                              2025 30                                                                       SerValAsnAsnSerGlnLeuValValSerValAlaGlyThrValGlu                              354045                                                                        GlyThrAsnGlnAspIleSerLeuLysPhePheGluIleAspLeuThr                              50 5560                                                                       SerArgProAlaHisGlyGlyLysThrGluGlnGlyLeuSerProLys                              65707580                                                                      SerLysProPheAlaThrAsp SerGlyAlaMetProHisLysLeuGlu                             859095                                                                        LysAlaAspLeuLeuLysAlaIleGlnGluGlnLeuIleAlaAsnVal                              100105 110                                                                    HisSerAsnAspAspTyrPheGluValIleAspPheAlaSerAspAla                              115120125                                                                     ThrIleThrAspArgAsnGlyLysValTyrPheAlaAspLysAsp Gly                             130135140                                                                     SerValThrLeuProThrGlnProValGlnGluPheLeuLeuSerGly                              145150155160                                                                  HisValArgV alArgProTyrLysGluLysProIleGlnAsnGlnAla                             165170175                                                                     LysSerValAspValGluTyrThrValGlnPheThrProLeuAsnPro                              180 185190                                                                    AspAspAspPheArgProGlyLeuLysAspThrLysLeuLeuLysThr                              195200205                                                                     LeuAlaIleGlyAspThrIleThrSerGlnGlu LeuLeuAlaGlnAla                             210215220                                                                     GlnSerIleLeuAsnLysThrHisProGlyTyrThrIleTyrGluArg                              225230235240                                                                  AspSerSerIleValThrHisAspAsnAspIlePheArgThrIleLeu                              245250255                                                                     ProMetAspGlnGluPheThrTyrHisValLysAsnArgGluGlnAla                               260265270                                                                    TyrGluIleAsnLysLysSerGlyLeuAsnGluGluIleAsnAsnThr                              275280285                                                                     AspLeuIleSerGluLysTyrT yrValLeuLysLysGlyGluLysPro                             290295300                                                                     TyrAspProPheAspArgSerHisLeuLysLeuPheThrIleLysTyr                              305310315 320                                                                 ValAspValAsnThrAsnGluLeuLeuLysSerGluGlnLeuLeuThr                              325330335                                                                     AlaSerGluArgAsnLeuAspPheArgAspLeuTyrAspPro ArgAsp                             340345350                                                                     LysAlaLysLeuLeuTyrAsnAsnLeuAspAlaPheGlyIleMetAsp                              355360365                                                                     TyrThrLeuTh rGlyLysValGluAspAsnHisAspAspThrAsnArg                             370375380                                                                     IleIleThrValTyrMetGlyLysArgProGluGlyGluAsnAlaSer                              385390 395400                                                                 TyrHisLeuAlaTyrAspLysAspArgTyrThrGluGluGluArgGlu                              405410415                                                                     ValTyrSerTyrLeuArgTyrThrGlyThr ProIleProAspAsnPro                             420425430                                                                     AsnAspLysValGluLeuGlnGlyValValProArgGlyIleAlaGly                              435440445                                                                     ProGluTrpLeuLeuAspArgProSerValAsnAsnSerGlnLeuVal                              450455460                                                                     ValSerValAlaGlyThrValGluGlyThrAsnGlnAspIleSerLeu                              465 470475480                                                                 LysPhePheGluIleAspLeuThrSerArgProAlaHisGlyGlyLys                              485490495                                                                     ThrGluGlnGlyLeuSerP roLysSerLysProPheAlaThrAspSer                             500505510                                                                     GlyAlaMetProHisLysLeuGluLysAlaAspLeuLeuLysAlaIle                              515520 525                                                                    GlnGluGlnLeuIleAlaAsnValHisSerAsnAspAspTyrPheGlu                              530535540                                                                     ValIleAspPheAlaSerAspAlaThrIleThrAspArgAsnGlyLys                              545 550555560                                                                 ValTyrPheAlaAspLysAspGlySerValThrLeuProThrGlnPro                              565570575                                                                     ValGlnGl uPheLeuLeuSerGlyHisValArgValArgProTyrLys                             580585590                                                                     GluLysProIleGlnAsnGlnAlaLysSerValAspValGluTyrThr                              595 600605                                                                    ValGlnPheThrProLeuAsnProAspAspAspPheArgProGlyLeu                              610615620                                                                     LysAspThrLysLeuLeuLysThrLeuAlaIleGlyAspT hrIleThr                             625630635640                                                                  SerGlnGluLeuLeuAlaGlnAlaGlnSerIleLeuAsnLysThrHis                              6456506 55                                                                    ProGlyTyrThrIleTyrGluArgAspSerSerIleValThrHisAsp                              660665670                                                                     AsnAspIlePheArgThrIleLeuProMetAspGlnGluPheThrTyr                               675680685                                                                    HisValLysAsnArgGluGlnAlaTyrGluIleAsnLysLysSerGly                              690695700                                                                     LeuAsnGluGluIleAsnAsnThrAspLe uIleSerGluLysTyrTyr                             705710715720                                                                  ValLeuLysLysGlyGluLysProTyrAspProPheAspArgSerHis                              725730 735                                                                    LeuLysLeuPheThrIleLysTyrValAspValAsnThrAsnGluLeu                              740745750                                                                     LeuLysSerGluGlnLeuLeuThrAlaSerGluArgAsnLeuA spPhe                             755760765                                                                     ArgAspLeuTyrAspProArgAspLysAlaLysLeuLeuTyrAsnAsn                              770775780                                                                     LeuAspAlaPheGlyIle MetAspTyrThrLeuThrGlyLysValGlu                             785790795800                                                                  AspAsnHisAspAspThrAsnArgIleIleThrValTyrMetGlyLys                              805 810815                                                                    ArgProGluGlyGluAsnAlaSerTyrHisLeuAlaTyrAspLysAsp                              820825830                                                                     ArgTyrThrGluGluGluArgGluValTyrSe rTyrLeuArgTyrThr                             835840845                                                                     GlyThrProIleProAspAsnProAsnAspLys                                             850855                                                                        (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..63                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2938"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GATAACCCTAACGACAAAGTAGAGCTGCAGGGAGTAGTTCCTCGTGG AATTGCTGGACCT60               GAG63                                                                         (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          ( ii) MOLECULE TYPE: cDNA                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2754"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GCTATCGGTGACACCAT17                                                           (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3639"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      GCTGCAGGGAGTAGTTC 17                                                          (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2253 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..2253                                                          (D) OTHER INFORMATION: /note=                                                "Met-corestreptokinase-corestreptokinase fusion                               linked by thrombin-cleavable VELQGVVPRG"                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 4..2244                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 4..2244                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      CATATGAGCCAATTAGTTG TTAGCGTTGCTGGTACTGTTGAGGGGACG48                           MetSerGlnLeuValValSerValAlaGlyThrValGluGlyThr                                 151015                                                                        AATCAAGACATTAGT CTTAAATTTTTTGAAATTGACCTAACATCACGA96                           AsnGlnAspIleSerLeuLysPhePheGluIleAspLeuThrSerArg                              202530                                                                        CCTGCTCATGGAGGA AAGACAGAGCAAGGCTTAAGTCCAAAATCAAAA144                          ProAlaHisGlyGlyLysThrGluGlnGlyLeuSerProLysSerLys                              354045                                                                        CCATTTGCTACTGATAG TGGCGCGATGCCACATAAACTTGAAAAAGCT192                          ProPheAlaThrAspSerGlyAlaMetProHisLysLeuGluLysAla                              505560                                                                        GACTTACTAAAGGCTATTCAAG AACAATTGATCGCTAACGTCCACAGT240                          AspLeuLeuLysAlaIleGlnGluGlnLeuIleAlaAsnValHisSer                              657075                                                                        AACGACGACTACTTTGAGGTCATTGATTTT GCAAGCGATGCAACCATT288                          AsnAspAspTyrPheGluValIleAspPheAlaSerAspAlaThrIle                              80859095                                                                      ACTGATCGAAACGGCAAGGTCTACTTT GCTGACAAAGATGGTTCGGTA336                          ThrAspArgAsnGlyLysValTyrPheAlaAspLysAspGlySerVal                              100105110                                                                     ACCTTGCCGACCCAACCTGTCCAAGA ATTTTTGCTAAGCGGACATGTG384                          ThrLeuProThrGlnProValGlnGluPheLeuLeuSerGlyHisVal                              115120125                                                                     CGCGTTAGACCATATAAAGAAAAACCAA TACAAAATCAAGCGAAATCT432                          ArgValArgProTyrLysGluLysProIleGlnAsnGlnAlaLysSer                              130135140                                                                     GTTGATGTGGAATATACTGTACAGTTTACTCCC TTAAACCCTGATGAC480                          ValAspValGluTyrThrValGlnPheThrProLeuAsnProAspAsp                              145150155                                                                     GATTTCAGACCAGGTCTCAAAGATACTAAGCTATTGAAAACA CTAGCT528                          AspPheArgProGlyLeuLysAspThrLysLeuLeuLysThrLeuAla                              160165170175                                                                  ATCGGTGACACCATCACATCTCAAGAATTACTAGCTCA AGCACAAAGC576                          IleGlyAspThrIleThrSerGlnGluLeuLeuAlaGlnAlaGlnSer                              180185190                                                                     ATTTTAAACAAAACCCATCCAGGCTATACGATTTATG AACGTGACTCC624                          IleLeuAsnLysThrHisProGlyTyrThrIleTyrGluArgAspSer                              195200205                                                                     TCAATCGTCACTCATGACAATGACATTTTCCGTACGATT TTACCAATG672                          SerIleValThrHisAspAsnAspIlePheArgThrIleLeuProMet                              210215220                                                                     GATCAAGAGTTTACTTACCATGTCAAAAATCGGGAACAAGCTTA TGAG720                          AspGlnGluPheThrTyrHisValLysAsnArgGluGlnAlaTyrGlu                              225230235                                                                     ATCAATAAAAAATCTGGTCTGAATGAAGAAATAAACAACACTGACCTG 768                          IleAsnLysLysSerGlyLeuAsnGluGluIleAsnAsnThrAspLeu                              240245250255                                                                  ATCTCTGAGAAATATTACGTCCTTAAAAAAGGGGAAAAGCCGTATGAT 816                          IleSerGluLysTyrTyrValLeuLysLysGlyGluLysProTyrAsp                              260265270                                                                     CCCTTTGATCGCAGTCACTTGAAACTGTTCACCATCAAATACGTTGAT 864                          ProPheAspArgSerHisLeuLysLeuPheThrIleLysTyrValAsp                              275280285                                                                     GTCAACACCAACGAATTGCTAAAAAGCGAGCAGCTCTTAACAGCTAGC 912                          ValAsnThrAsnGluLeuLeuLysSerGluGlnLeuLeuThrAlaSer                              290295300                                                                     GAACGTAACTTAGACTTCAGAGATTTATACGATCCTCGTGATAAGGCT960                            GluArgAsnLeuAspPheArgAspLeuTyrAspProArgAspLysAla                             305310315                                                                     AAACTACTCTACAACAATCTCGATGCTTTTGGTATTATGGACTATACC1008                          LysLeuLe uTyrAsnAsnLeuAspAlaPheGlyIleMetAspTyrThr                             320325330335                                                                  TTAACTGGAAAAGTAGAAGATAATCACGATGACACCAACCGTATCATA1056                          LeuT hrGlyLysValGluAspAsnHisAspAspThrAsnArgIleIle                             340345350                                                                     ACCGTTTATATGGGCAAGCGACCCGAAGGAGAGAATGCTAGCTATCAT1104                          Thr ValTyrMetGlyLysArgProGluGlyGluAsnAlaSerTyrHis                             355360365                                                                     TTAGCCGTAGAGCTGCAGGGAGTAGTTCCTCGTGGAAGCCAATTAGTT1152                          LeuAla ValGluLeuGlnGlyValValProArgGlySerGlnLeuVal                             370375380                                                                     GTTAGCGTTGCTGGTACTGTTGAGGGGACGAATCAAGACATTAGTCTT1200                          ValSerValAl aGlyThrValGluGlyThrAsnGlnAspIleSerLeu                             385390395                                                                     AAATTTTTTGAAATTGACCTAACATCACGACCTGCTCATGGAGGAAAG1248                          LysPhePheGluIleAspL euThrSerArgProAlaHisGlyGlyLys                             400405410415                                                                  ACAGAGCAAGGCTTAAGTCCAAAATCAAAACCATTTGCTACTGATAGT1296                          ThrGluGlnGlyLeu SerProLysSerLysProPheAlaThrAspSer                             420425430                                                                     GGCGCGATGCCACATAAACTTGAAAAAGCTGACTTACTAAAGGCTATT1344                          GlyAlaMetProHi sLysLeuGluLysAlaAspLeuLeuLysAlaIle                             435440445                                                                     CAAGAACAATTGATCGCTAACGTCCACAGTAACGACGACTACTTTGAG1392                          GlnGluGlnLeuIleA laAsnValHisSerAsnAspAspTyrPheGlu                             450455460                                                                     GTCATTGATTTTGCAAGCGATGCAACCATTACTGATCGAAACGGCAAG1440                          ValIleAspPheAlaSerAsp AlaThrIleThrAspArgAsnGlyLys                             465470475                                                                     GTCTACTTTGCTGACAAAGATGGTTCGGTAACCTTGCCGACCCAACCT1488                          ValTyrPheAlaAspLysAspGlySerVal ThrLeuProThrGlnPro                             480485490495                                                                  GTCCAAGAATTTTTGCTAAGCGGACATGTGCGCGTTAGACCATATAAA1536                          ValGlnGluPheLeuLeuSerGlyHi sValArgValArgProTyrLys                             500505510                                                                     GAAAAACCAATACAAAATCAAGCGAAATCTGTTGATGTGGAATATACT1584                          GluLysProIleGlnAsnGlnAlaL ysSerValAspValGluTyrThr                             515520525                                                                     GTACAGTTTACTCCCTTAAACCCTGATGACGATTTCAGACCAGGTCTC1632                          ValGlnPheThrProLeuAsnProAsp AspAspPheArgProGlyLeu                             530535540                                                                     AAAGATACTAAGCTATTGAAAACACTAGCTATCGGTGACACCATCACA1680                          LysAspThrLysLeuLeuLysThrLeuAlaIle GlyAspThrIleThr                             545550555                                                                     TCTCAAGAATTACTAGCTCAAGCACAAAGCATTTTAAACAAAACCCAT1728                          SerGlnGluLeuLeuAlaGlnAlaGlnSerIleLeuAsnLy sThrHis                             560565570575                                                                  CCAGGCTATACGATTTATGAACGTGACTCCTCAATCGTCACTCATGAC1776                          ProGlyTyrThrIleTyrGluArgAspSerSerIleV alThrHisAsp                             580585590                                                                     AATGACATTTTCCGTACGATTTTACCAATGGATCAAGAGTTTACTTAC1824                          AsnAspIlePheArgThrIleLeuProMetAspGln GluPheThrTyr                             595600605                                                                     CATGTCAAAAATCGGGAACAAGCTTATGAGATCAATAAAAAATCTGGT1872                          HisValLysAsnArgGluGlnAlaTyrGluIleAsnLys LysSerGly                             610615620                                                                     CTGAATGAAGAAATAAACAACACTGACCTGATCTCTGAGAAATATTAC1920                          LeuAsnGluGluIleAsnAsnThrAspLeuIleSerGluLysTy rTyr                             625630635                                                                     GTCCTTAAAAAAGGGGAAAAGCCGTATGATCCCTTTGATCGCAGTCAC1968                          ValLeuLysLysGlyGluLysProTyrAspProPheAspArgSerHis                              640 645650655                                                                 TTGAAACTGTTCACCATCAAATACGTTGATGTCAACACCAACGAATTG2016                          LeuLysLeuPheThrIleLysTyrValAspValAsnThrAsnGluLeu                              660665670                                                                     CTAAAAAGCGAGCAGCTCTTAACAGCTAGCGAACGTAACTTAGACTTC2064                          LeuLysSerGluGlnLeuLeuThrAlaSerGluArgAsnLeuAspPh e                             675680685                                                                     AGAGATTTATACGATCCTCGTGATAAGGCTAAACTACTCTACAACAAT2112                          ArgAspLeuTyrAspProArgAspLysAlaLysLeuLeuTyrAsnAsn                               690695700                                                                    CTCGATGCTTTTGGTATTATGGACTATACCTTAACTGGAAAAGTAGAA2160                          LeuAspAlaPheGlyIleMetAspTyrThrLeuThrGlyLysValGlu                              705 710715                                                                    GATAATCACGATGACACCAACCGTATCATAACCGTTTATATGGGCAAG2208                          AspAsnHisAspAspThrAsnArgIleIleThrValTyrMetGlyLys                              720 725730735                                                                 CGACCCGAAGGAGAGAATGCTAGCTATCATTTAGCCTAAGGATCC2253                             ArgProGluGlyGluAsnAlaSerTyrHisLeuAla                                          740 745                                                                       (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 747 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      MetSerGlnLeuValValSerValAlaGlyThrValGluGlyThrAsn                              1 51015                                                                       GlnAspIleSerLeuLysPhePheGluIleAspLeuThrSerArgPro                              202530                                                                        AlaHisGlyGlyLysThrGlu GlnGlyLeuSerProLysSerLysPro                             354045                                                                        PheAlaThrAspSerGlyAlaMetProHisLysLeuGluLysAlaAsp                              5055 60                                                                       LeuLeuLysAlaIleGlnGluGlnLeuIleAlaAsnValHisSerAsn                              65707580                                                                      AspAspTyrPheGluValIleAspPheAlaSerAspAlaThrIleThr                              859095                                                                        AspArgAsnGlyLysValTyrPheAlaAspLysAspGlySerValThr                              100105110                                                                     LeuProThrG lnProValGlnGluPheLeuLeuSerGlyHisValArg                             115120125                                                                     ValArgProTyrLysGluLysProIleGlnAsnGlnAlaLysSerVal                              130135 140                                                                    AspValGluTyrThrValGlnPheThrProLeuAsnProAspAspAsp                              145150155160                                                                  PheArgProGlyLeuLysAspThrLysLeuLeuLys ThrLeuAlaIle                             165170175                                                                     GlyAspThrIleThrSerGlnGluLeuLeuAlaGlnAlaGlnSerIle                              180185190                                                                     LeuAsnLysThrHisProGlyTyrThrIleTyrGluArgAspSerSer                              195200205                                                                     IleValThrHisAspAsnAspIlePheArgThrIleLeuProMetAsp                              210 215220                                                                    GlnGluPheThrTyrHisValLysAsnArgGluGlnAlaTyrGluIle                              225230235240                                                                  AsnLysLysSerGlyLeuAsnGluG luIleAsnAsnThrAspLeuIle                             245250255                                                                     SerGluLysTyrTyrValLeuLysLysGlyGluLysProTyrAspPro                              260265 270                                                                    PheAspArgSerHisLeuLysLeuPheThrIleLysTyrValAspVal                              275280285                                                                     AsnThrAsnGluLeuLeuLysSerGluGlnLeuLeuThrAlaSerGlu                               290295300                                                                    ArgAsnLeuAspPheArgAspLeuTyrAspProArgAspLysAlaLys                              305310315320                                                                  LeuLeuTyrAsnAs nLeuAspAlaPheGlyIleMetAspTyrThrLeu                             325330335                                                                     ThrGlyLysValGluAspAsnHisAspAspThrAsnArgIleIleThr                              340 345350                                                                    ValTyrMetGlyLysArgProGluGlyGluAsnAlaSerTyrHisLeu                              355360365                                                                     AlaValGluLeuGlnGlyValValProArgGlySer GlnLeuValVal                             370375380                                                                     SerValAlaGlyThrValGluGlyThrAsnGlnAspIleSerLeuLys                              385390395400                                                                  Ph ePheGluIleAspLeuThrSerArgProAlaHisGlyGlyLysThr                             405410415                                                                     GluGlnGlyLeuSerProLysSerLysProPheAlaThrAspSerGly                               420425430                                                                    AlaMetProHisLysLeuGluLysAlaAspLeuLeuLysAlaIleGln                              435440445                                                                     GluGlnLeuIleAlaAsnValHisS erAsnAspAspTyrPheGluVal                             450455460                                                                     IleAspPheAlaSerAspAlaThrIleThrAspArgAsnGlyLysVal                              465470475 480                                                                 TyrPheAlaAspLysAspGlySerValThrLeuProThrGlnProVal                              485490495                                                                     GlnGluPheLeuLeuSerGlyHisValArgValArgProTyrLys Glu                             500505510                                                                     LysProIleGlnAsnGlnAlaLysSerValAspValGluTyrThrVal                              515520525                                                                     GlnPheThrProLe uAsnProAspAspAspPheArgProGlyLeuLys                             530535540                                                                     AspThrLysLeuLeuLysThrLeuAlaIleGlyAspThrIleThrSer                              545550 555560                                                                 GlnGluLeuLeuAlaGlnAlaGlnSerIleLeuAsnLysThrHisPro                              565570575                                                                     GlyTyrThrIleTyrGluArgAspSerSerIleV alThrHisAspAsn                             580585590                                                                     AspIlePheArgThrIleLeuProMetAspGlnGluPheThrTyrHis                              595600605                                                                     Val LysAsnArgGluGlnAlaTyrGluIleAsnLysLysSerGlyLeu                             610615620                                                                     AsnGluGluIleAsnAsnThrAspLeuIleSerGluLysTyrTyrVal                              625630 635640                                                                 LeuLysLysGlyGluLysProTyrAspProPheAspArgSerHisLeu                              645650655                                                                     LysLeuPheThrIleLysTyrVa lAspValAsnThrAsnGluLeuLeu                             660665670                                                                     LysSerGluGlnLeuLeuThrAlaSerGluArgAsnLeuAspPheArg                              675680 685                                                                    AspLeuTyrAspProArgAspLysAlaLysLeuLeuTyrAsnAsnLeu                              690695700                                                                     AspAlaPheGlyIleMetAspTyrThrLeuThrGlyLysValGluAsp                              705 710715720                                                                 AsnHisAspAspThrAsnArgIleIleThrValTyrMetGlyLysArg                              725730735                                                                     ProGluGlyGlu AsnAlaSerTyrHisLeuAla                                            740745                                                                        (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 ( A) NAME/KEY: misc.sub.-- feature                                            (B) LOCATION: 1..61                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3861"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      GCTATCATTTAGCCGTAGAGCTGCAGGGAGTAGTTCCTCGTGGAAGCCAATTAGTTGTTA60                G 61                                                                          (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1458 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..1458                                                         (D) OTHER INFORMATION: /note="Hirudin-streptokinase                            fusion linked by Factor Xa cleavable IEGR"                                   (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1449                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..1449                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GTTGTTTACACCGACTGTACTGAATCCGGACAAAACCTGTGTTTGTGT 48                           ValValTyrThrAspCysThrGluSerGlyGlnAsnLeuCysLeuCys                              151015                                                                        GAGGGTTCTAACGTCTGTGGTCAGGGTAACAAATGCATCCTGGGTTCC 96                           GluGlySerAsnValCysGlyGlnGlyAsnLysCysIleLeuGlySer                              202530                                                                        GACGGTGAAAAGAACCAATGTGTCACTGGTGAAGGTACCCCAAAGCCG144                           AspGlyGluLysAsnGlnCysValThrGlyGluGlyThrProLysPro                              354045                                                                        CAGTCCCACAACGATGGAGATTTCGAAGAAATCCCAGAAGAATATCTG192                           GlnS erHisAsnAspGlyAspPheGluGluIleProGluGluTyrLeu                             505560                                                                        CAGATCGAAGGTAGAATTGCTGGACCTGAGTGGCTGCTAGACCGTCCA240                           GlnIleGluGly ArgIleAlaGlyProGluTrpLeuLeuAspArgPro                             65707580                                                                      TCTGTCAACAACAGCCAATTAGTTGTTAGCGTTGCTGGTACTGTTGAG288                           SerValAsn AsnSerGlnLeuValValSerValAlaGlyThrValGlu                             859095                                                                        GGGACGAATCAAGACATTAGTCTTAAATTTTTTGAAATTGACCTAACA336                           GlyThrAs nGlnAspIleSerLeuLysPhePheGluIleAspLeuThr                             100105110                                                                     TCACGACCTGCTCATGGAGGAAAGACAGAGCAAGGCTTAAGTCCAAAA384                           SerArgPro AlaHisGlyGlyLysThrGluGlnGlyLeuSerProLys                             115120125                                                                     TCAAAACCATTTGCTACTGATAGTGGCGCGATGCCACATAAACTTGAA432                           SerLysProPheAla ThrAspSerGlyAlaMetProHisLysLeuGlu                             130135140                                                                     AAAGCTGACTTACTAAAGGCTATTCAAGAACAATTGATCGCTAACGTC480                           LysAlaAspLeuLeuLysAlaIl eGlnGluGlnLeuIleAlaAsnVal                             145150155160                                                                  CACAGTAACGACGACTACTTTGAGGTCATTGATTTTGCAAGCGATGCA528                           HisSerAsnAspAspTyrP heGluValIleAspPheAlaSerAspAla                             165170175                                                                     ACCATTACTGATCGAAACGGCAAGGTCTACTTTGCTGACAAAGATGGT576                           ThrIleThrAspArgAsn GlyLysValTyrPheAlaAspLysAspGly                             180185190                                                                     TCGGTAACCTTGCCGACCCAACCTGTCCAAGAATTTTTGCTAAGCGGA624                           SerValThrLeuProThrGln ProValGlnGluPheLeuLeuSerGly                             195200205                                                                     CATGTGCGCGTTAGACCATATAAAGAAAAACCAATACAAAATCAAGCG672                           HisValArgValArgProTyrLysGl uLysProIleGlnAsnGlnAla                             210215220                                                                     AAATCTGTTGATGTGGAATATACTGTACAGTTTACTCCCTTAAACCCT720                           LysSerValAspValGluTyrThrValGlnPheT hrProLeuAsnPro                             225230235240                                                                  GATGACGATTTCAGACCAGGTCTCAAAGATACTAAGCTATTGAAAACA768                           AspAspAspPheArgProGlyLeuLysAsp ThrLysLeuLeuLysThr                             245250255                                                                     CTAGCTATCGGTGACACCATCACATCTCAAGAATTACTAGCTCAAGCA816                           LeuAlaIleGlyAspThrIleThrSerGln GluLeuLeuAlaGlnAla                             260265270                                                                     CAAAGCATTTTAAACAAAACCCATCCAGGCTATACGATTTATGAACGT864                           GlnSerIleLeuAsnLysThrHisProGlyTy rThrIleTyrGluArg                             275280285                                                                     GACTCCTCAATCGTCACTCATGACAATGACATTTTCCGTACGATTTTA912                           AspSerSerIleValThrHisAspAsnAspIlePheA rgThrIleLeu                             290295300                                                                     CCAATGGATCAAGAGTTTACTTACCATGTCAAAAATCGGGAACAAGCT960                           ProMetAspGlnGluPheThrTyrHisValLysAsnArgGluGln Ala                             305310315320                                                                  TATGAGATCAATAAAAAATCTGGTCTGAATGAAGAAATAAACAACACT1008                          TyrGluIleAsnLysLysSerGlyLeuAsnGluGluIleAsn AsnThr                             325330335                                                                     GACCTGATCTCTGAGAAATATTACGTCCTTAAAAAAGGGGAAAAGCCG1056                          AspLeuIleSerGluLysTyrTyrValLeuLysLysGlyGl uLysPro                             340345350                                                                     TATGATCCCTTTGATCGCAGTCACTTGAAACTGTTCACCATCAAATAC1104                          TyrAspProPheAspArgSerHisLeuLysLeuPheThrIleL ysTyr                             355360365                                                                     GTTGATGTCAACACCAACGAATTGCTAAAAAGCGAGCAGCTCTTAACA1152                          ValAspValAsnThrAsnGluLeuLeuLysSerGluGlnLeuLeuThr                               370375380                                                                    GCTAGCGAACGTAACTTAGACTTCAGAGATTTATACGATCCTCGTGAT1200                          AlaSerGluArgAsnLeuAspPheArgAspLeuTyrAspProArgAsp                              385 390395400                                                                 AAGGCTAAACTACTCTACAACAATCTCGATGCTTTTGGTATTATGGAC1248                          LysAlaLysLeuLeuTyrAsnAsnLeuAspAlaPheGlyIleMetAsp                               405410415                                                                    TATACCTTAACTGGAAAAGTAGAAGATAATCACGATGACACCAACCGT1296                          TyrThrLeuThrGlyLysValGluAspAsnHisAspAspThrAsnArg                               420425430                                                                    ATCATAACCGTTTATATGGGCAAGCGACCCGAAGGAGAGAATGCTAGC1344                          IleIleThrValTyrMetGlyLysArgProGluGlyGluAsnAlaSer                               435440445                                                                    TATCATTTAGCCTATGATAAAGATCGTTATACCGAAGAAGAACGAGAA1392                          TyrHisLeuAlaTyrAspLysAspArgTyrThrGluGluGluArgGlu                              450 455460                                                                    GTTTACAGCTACCTGCGTTATACAGGGACACCTATACCTGATAACCCT1440                          ValTyrSerTyrLeuArgTyrThrGlyThrProIleProAspAsnPro                              465470 475480                                                                 AACGACAAATAAGGATCC1458                                                        AsnAspLys                                                                     (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 483 amino acids                                                   (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      ValValTyrThrAspCysThrGluSerGlyGlnAsnLeuCysLeuCys                              151015                                                                        GluGlySerAsnValCysGlyGlnGly AsnLysCysIleLeuGlySer                             202530                                                                        AspGlyGluLysAsnGlnCysValThrGlyGluGlyThrProLysPro                              3540 45                                                                       GlnSerHisAsnAspGlyAspPheGluGluIleProGluGluTyrLeu                              505560                                                                        GlnIleGluGlyArgIleAlaGlyProGluTrpLeuLeuAspArgPro                              65 707580                                                                     SerValAsnAsnSerGlnLeuValValSerValAlaGlyThrValGlu                              859095                                                                        GlyThrAsnGlnAsp IleSerLeuLysPhePheGluIleAspLeuThr                             100105110                                                                     SerArgProAlaHisGlyGlyLysThrGluGlnGlyLeuSerProLys                              115120 125                                                                    SerLysProPheAlaThrAspSerGlyAlaMetProHisLysLeuGlu                              130135140                                                                     LysAlaAspLeuLeuLysAlaIleGlnGluGlnLeuIleAlaAsnVal                              145150155160                                                                  HisSerAsnAspAspTyrPheGluValIleAspPheAlaSerAspAla                              165170175                                                                     ThrI leThrAspArgAsnGlyLysValTyrPheAlaAspLysAspGly                             180185190                                                                     SerValThrLeuProThrGlnProValGlnGluPheLeuLeuSerGly                              195 200205                                                                    HisValArgValArgProTyrLysGluLysProIleGlnAsnGlnAla                              210215220                                                                     LysSerValAspValGluTyrThrValGlnPheThr ProLeuAsnPro                             225230235240                                                                  AspAspAspPheArgProGlyLeuLysAspThrLysLeuLeuLysThr                              245250 255                                                                    LeuAlaIleGlyAspThrIleThrSerGlnGluLeuLeuAlaGlnAla                              260265270                                                                     GlnSerIleLeuAsnLysThrHisProGlyTyrThrIleTyrGluArg                               275280285                                                                    AspSerSerIleValThrHisAspAsnAspIlePheArgThrIleLeu                              290295300                                                                     ProMetAspGlnGluPheThrTyrH isValLysAsnArgGluGlnAla                             305310315320                                                                  TyrGluIleAsnLysLysSerGlyLeuAsnGluGluIleAsnAsnThr                              3253 30335                                                                    AspLeuIleSerGluLysTyrTyrValLeuLysLysGlyGluLysPro                              340345350                                                                     TyrAspProPheAspArgSerHisLeuLysLeuPheThr IleLysTyr                             355360365                                                                     ValAspValAsnThrAsnGluLeuLeuLysSerGluGlnLeuLeuThr                              370375380                                                                     AlaSerGluArgAs nLeuAspPheArgAspLeuTyrAspProArgAsp                             385390395400                                                                  LysAlaLysLeuLeuTyrAsnAsnLeuAspAlaPheGlyIleMetAsp                              405 410415                                                                    TyrThrLeuThrGlyLysValGluAspAsnHisAspAspThrAsnArg                              420425430                                                                     IleIleThrValTyrMetGlyLysArgP roGluGlyGluAsnAlaSer                             435440445                                                                     TyrHisLeuAlaTyrAspLysAspArgTyrThrGluGluGluArgGlu                              450455460                                                                     Val TyrSerTyrLeuArgTyrThrGlyThrProIleProAspAsnPro                             465470475480                                                                  AsnAspLys                                                                     (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..46                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3317"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      CACTCAGGTCCAGCAATTCTACCTTCGATCTGCAGATATTCTTCTG 46                             (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: /note="oligonucleotiede BB3510"                        ( xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                     CACTATCAGTAGCAAAT17                                                           (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1467 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..1467                                                         (D) OTHER INFORMATION: /note="Streptokinase-hirudin                           fusion linked by Factor Xa-cleavable IEGR"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1449                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..1449                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                       ATTGCTGGACCTGAGTGGCTGCTAGACCGTCCATCTGTCAACAACAGC48                           IleAlaGlyProGluTrpLeuLeuAspArgProSerValAsnAsnSer                              151015                                                                         CAATTAGTTGTTAGCGTTGCTGGTACTGTTGAGGGGACGAATCAAGAC96                           GlnLeuValValSerValAlaGlyThrValGluGlyThrAsnGlnAsp                              202530                                                                        AT TAGTCTTAAATTTTTTGAAATTGACCTAACATCACGACCTGCTCAT144                          IleSerLeuLysPhePheGluIleAspLeuThrSerArgProAlaHis                              354045                                                                        GGAGGAA AGACAGAGCAAGGCTTAAGTCCAAAATCAAAACCATTTGCT192                          GlyGlyLysThrGluGlnGlyLeuSerProLysSerLysProPheAla                              505560                                                                        ACTGATAGTGGCGCG ATGCCACATAAACTTGAAAAAGCTGACTTACTA240                          ThrAspSerGlyAlaMetProHisLysLeuGluLysAlaAspLeuLeu                              65707580                                                                      AAGGCTATTCAA GAACAATTGATCGCTAACGTCCACAGTAACGACGAC288                          LysAlaIleGlnGluGlnLeuIleAlaAsnValHisSerAsnAspAsp                              859095                                                                        TACTTTGAGGT CATTGATTTTGCAAGCGATGCAACCATTACTGATCGA336                          TyrPheGluValIleAspPheAlaSerAspAlaThrIleThrAspArg                              100105110                                                                     AACGGCAAGGTCT ACTTTGCTGACAAAGATGGTTCGGTAACCTTGCCG384                          AsnGlyLysValTyrPheAlaAspLysAspGlySerValThrLeuPro                              115120125                                                                     ACCCAACCTGTCCAAGAA TTTTTGCTAAGCGGACATGTGCGCGTTAGA432                          ThrGlnProValGlnGluPheLeuLeuSerGlyHisValArgValArg                              130135140                                                                     CCATATAAAGAAAAACCAATACAAAAT CAAGCGAAATCTGTTGATGTG480                          ProTyrLysGluLysProIleGlnAsnGlnAlaLysSerValAspVal                              145150155160                                                                  GAATATACTGTACAGTTTACTCC CTTAAACCCTGATGACGATTTCAGA528                          GluTyrThrValGlnPheThrProLeuAsnProAspAspAspPheArg                              165170175                                                                     CCAGGTCTCAAAGATACTAAGC TATTGAAAACACTAGCTATCGGTGAC576                          ProGlyLeuLysAspThrLysLeuLeuLysThrLeuAlaIleGlyAsp                              180185190                                                                     ACCATCACATCTCAAGAATTACTA GCTCAAGCACAAAGCATTTTAAAC624                          ThrIleThrSerGlnGluLeuLeuAlaGlnAlaGlnSerIleLeuAsn                              195200205                                                                     AAAACCCATCCAGGCTATACGATTTATGAA CGTGACTCCTCAATCGTC672                          LysThrHisProGlyTyrThrIleTyrGluArgAspSerSerIleVal                              210215220                                                                     ACTCATGACAATGACATTTTCCGTACGATTTTACCAAT GGATCAAGAG720                          ThrHisAspAsnAspIlePheArgThrIleLeuProMetAspGlnGlu                              225230235240                                                                  TTTACTTACCATGTCAAAAATCGGGAACAAGCTT ATGAGATCAATAAA768                          PheThrTyrHisValLysAsnArgGluGlnAlaTyrGluIleAsnLys                              245250255                                                                     AAATCTGGTCTGAATGAAGAAATAAACAACACT GACCTGATCTCTGAG816                          LysSerGlyLeuAsnGluGluIleAsnAsnThrAspLeuIleSerGlu                              260265270                                                                     AAATATTACGTCCTTAAAAAAGGGGAAAAGCCGTA TGATCCCTTTGAT864                          LysTyrTyrValLeuLysLysGlyGluLysProTyrAspProPheAsp                              275280285                                                                     CGCAGTCACTTGAAACTGTTCACCATCAAATACGTTGATG TCAACACC912                          ArgSerHisLeuLysLeuPheThrIleLysTyrValAspValAsnThr                              290295300                                                                     AACGAATTGCTAAAAAGCGAGCAGCTCTTAACAGCTAGCGAACGTAAC 960                          AsnGluLeuLeuLysSerGluGlnLeuLeuThrAlaSerGluArgAsn                              305310315320                                                                  TTAGACTTCAGAGATTTATACGATCCTCGTGATAAGGCTAAACTA CTC1008                         LeuAspPheArgAspLeuTyrAspProArgAspLysAlaLysLeuLeu                              325330335                                                                     TACAACAATCTCGATGCTTTTGGTATTATGGACTATACCTTAAC TGGA1056                         TyrAsnAsnLeuAspAlaPheGlyIleMetAspTyrThrLeuThrGly                              340345350                                                                     AAAGTAGAAGATAATCACGATGACACCAACCGTATCATAACCGTTT AT1104                         LysValGluAspAsnHisAspAspThrAsnArgIleIleThrValTyr                              355360365                                                                     ATGGGCAAGCGACCCGAAGGAGAGAATGCTAGCTATCATTTAGCCTAT 1152                         MetGlyLysArgProGluGlyGluAsnAlaSerTyrHisLeuAlaTyr                              370375380                                                                     GATAAAGATCGTTATACCGAAGAAGAACGAGAAGTTTACAGCTACCTG1200                          AspL ysAspArgTyrThrGluGluGluArgGluValTyrSerTyrLeu                             385390395400                                                                  CGTTATACAGGGACACCTATACCTGATAACCCTAACGACAAAATCGAA1248                           ArgTyrThrGlyThrProIleProAspAsnProAsnAspLysIleGlu                             405410415                                                                     GGTAGAGTTGTTTACACCGACTGTACTGAATCCGGACAAAACCTGTGT1296                           GlyArgValValTyrThrAspCysThrGluSerGlyGlnAsnLeuCys                             420425430                                                                     TTGTGTGAGGGTTCTAACGTCTGTGGTCAGGGTAACAAATGCATCCTG1344                          Le uCysGluGlySerAsnValCysGlyGlnGlyAsnLysCysIleLeu                             435440445                                                                     GGTTCCGACGGTGAAAAGAACCAATGTGTCACTGGTGAAGGTACCCCA1392                          GlySerA spGlyGluLysAsnGlnCysValThrGlyGluGlyThrPro                             450455460                                                                     AAGCCGCAGTCCCACAACGATGGAGATTTCGAAGAAATCCCAGAAGAA1440                          LysProGlnSerHis AsnAspGlyAspPheGluGluIleProGluGlu                             465470475480                                                                  TATCTGCAGTAATAGGGATCCGAATTC1467                                               TyrLeuGln                                                                      (2) INFORMATION FOR SEQ ID NO:47:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 483 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      IleAlaGlyProGluTrpLeuLeuAspArgProSerValAsnAsnSer                              15 1015                                                                       GlnLeuValValSerValAlaGlyThrValGluGlyThrAsnGlnAsp                              202530                                                                        IleSerLeuLysPhePheGluIleAspLeuTh rSerArgProAlaHis                             354045                                                                        GlyGlyLysThrGluGlnGlyLeuSerProLysSerLysProPheAla                              505560                                                                        ThrAsp SerGlyAlaMetProHisLysLeuGluLysAlaAspLeuLeu                             65707580                                                                      LysAlaIleGlnGluGlnLeuIleAlaAsnValHisSerAsnAspAsp                               859095                                                                       TyrPheGluValIleAspPheAlaSerAspAlaThrIleThrAspArg                              100105110                                                                     AsnGlyLysValTyrPheAla AspLysAspGlySerValThrLeuPro                             115120125                                                                     ThrGlnProValGlnGluPheLeuLeuSerGlyHisValArgValArg                              130135 140                                                                    ProTyrLysGluLysProIleGlnAsnGlnAlaLysSerValAspVal                              145150155160                                                                  GluTyrThrValGlnPheThrProLeuAsnProAspAspAspPheAr g                             165170175                                                                     ProGlyLeuLysAspThrLysLeuLeuLysThrLeuAlaIleGlyAsp                              180185190                                                                     ThrIleThr SerGlnGluLeuLeuAlaGlnAlaGlnSerIleLeuAsn                             195200205                                                                     LysThrHisProGlyTyrThrIleTyrGluArgAspSerSerIleVal                              210215 220                                                                    ThrHisAspAsnAspIlePheArgThrIleLeuProMetAspGlnGlu                              225230235240                                                                  PheThrTyrHisValLysAsnArgGluGlnAlaTy rGluIleAsnLys                             245250255                                                                     LysSerGlyLeuAsnGluGluIleAsnAsnThrAspLeuIleSerGlu                              26026527 0                                                                    LysTyrTyrValLeuLysLysGlyGluLysProTyrAspProPheAsp                              275280285                                                                     ArgSerHisLeuLysLeuPheThrIleLysTyrValAspValAsnThr                              290 295300                                                                    AsnGluLeuLeuLysSerGluGlnLeuLeuThrAlaSerGluArgAsn                              305310315320                                                                  LeuAspPheArgAspLeuTyrAsp ProArgAspLysAlaLysLeuLeu                             325330335                                                                     TyrAsnAsnLeuAspAlaPheGlyIleMetAspTyrThrLeuThrGly                              340345 350                                                                    LysValGluAspAsnHisAspAspThrAsnArgIleIleThrValTyr                              355360365                                                                     MetGlyLysArgProGluGlyGluAsnAlaSerTyrHisLeuAlaTy r                             370375380                                                                     AspLysAspArgTyrThrGluGluGluArgGluValTyrSerTyrLeu                              385390395400                                                                  ArgTyrThrGly ThrProIleProAspAsnProAsnAspLysIleGlu                             405410415                                                                     GlyArgValValTyrThrAspCysThrGluSerGlyGlnAsnLeuCys                              420 425430                                                                    LeuCysGluGlySerAsnValCysGlyGlnGlyAsnLysCysIleLeu                              435440445                                                                     GlySerAspGlyGluLysAsnGlnCysValThrGly GluGlyThrPro                             450455460                                                                     LysProGlnSerHisAsnAspGlyAspPheGluGluIleProGluGlu                              465470475480                                                                  T yrLeuGln                                                                    (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..47                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3318"                         (x i) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                     TCGGTGTAAACAACTCTTCTACCTTCGATTTTGTCGTTAGGGTTATC47                             (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      ( ix) FEATURE:                                                                (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3623"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      GTGTAAACAACTCTACCTTCG21                                                       (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..41                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2011"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      AGCTTACCTGCCATGGTTGTTTACACCGACTGTACTGAATC 41                                  (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..44                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2012"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      TTGTCCGGATTCAGTACAGTCGGTGTAAACAACCATGGCAGGTA44                                (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..37                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2013"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      CGGACAAAACCTGTGTTTGTGTGAGGGTTCTAACGTC37                                       (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..37                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2014"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      GACCACAGACGTTAGAACCCTCACACAA ACACAGGTT37                                      (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..40                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2015"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      TGTGGTCAGGGTAACAAATGCATCCTGGGTTCCGACGGTG40                                    (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..40                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2016"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      TTCTTTTCACCGTCGGAACCCAGGATGCATTTGTTACCCT40                                    (2) INFORMATION FOR SEQ ID NO:56:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..35                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2017"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      AAAAGAACCAATG TGTCACTGGTGAAGGTACCCCA35                                        (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..35                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2018"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GCGGCTTTGGGGTACCTTCACCAGTGACACATTGG35                                         (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..39                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2019"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      AAGCCGCAGTCCCACAACGATGGAGATTTCGAAGAAATC 39                                    (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..39                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2020"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      CTTCTGGGATTTCTTCGAAATCTCCATCGTTGTGGGACT39                                     (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                            (B) LOCATION: 1..31                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2021"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      CCAGAAGAATATCTGCAGTAATAGGGATCCG31                                             (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..28                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2022"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      AATTCGGATCCCTATTACTGCAGATATT 28                                               (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2136"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      TGGTCTAACGCGCACT16                                                            (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3509"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      GAGTAAACTGTACAGTA17                                                           (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 ( A) LENGTH: 17 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3508"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      GATCTCATAAGCTTGTT 17                                                          (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..17                                                            (D) OTHER INFORMATION: /note="oligonucleotide BB2135"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      TTTAGCCTTATCACGAG17                                                           (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB3718"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      CGTTGATGTCAACACCA17                                                           (2) INFORMATION FOR SEQ ID NO:67:                                             (i ) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: /note="oligonucleotide BB2755"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      GACGACTACTTTGAGGT 17                                                          (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..17                                                          (D) OTHER INFORMATION: /note="oligonucleotide BB2134"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      CCCAACCTGTCCAAGAA17                                                           (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note="thrombin cleavable linker"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      ValGluLeuGlnGlyValValProArgGly10                                              15 10                                                                         (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note="N-terminal amino acids of                        native hirudin"                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      ValValTyrThrAsp5                                                              15                                                                            (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: /note="Factor Xa cleavable                             linker"                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      IleGluGlyArg4                                                                 ( 2) INFORMATION FOR SEQ ID NO:72:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..2                                                            (D) OTHER INFORMATION: /note="each is independently                           a hydrobphobic residue"                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                            (B) LOCATION: 5..6                                                            (D) OTHER INFORMATION: /note="each is independently                           a non-acidic residue"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      XaaXaaProArgXaaXaa6                                                           15                                                                            (2) INFORMATION FOR SEQ ID NO:73:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..2                                                            (D) OTHER INFORMATION: /note="The first amino acid is                         a hydrobphobic residue and the second is an acidic                            residue."                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                     XaaXaaGlyArg4                                                                 1                                                                         

We claim:
 1. A non-naturally occurring fusion protein suitable for useas a selectively acting fibrinolytic or anti-thrombotic agent comprisinga first sequence, a second sequence, and a Factor Xa cleavable linkersequence, wherein the Factor Xa cleavable linker sequence is IEGR (SEQID: NO: 71) and the first sequence and second sequence are chosen fromthe group consisting of streptokinase, hirudin and their conservativelysubstituted analogues, provided that when either the first or secondsequence is hirudin or its conservatively substituted analogue the otheris streptokinase or its conservatively substituted analogue.